3D-KompressionFH Giessen-Friedberg 3D-Kompression Multimedia-Seminar, WS 2001/2002 Michael Weyel.
Surface binding autoantibodies and their functional...
Transcript of Surface binding autoantibodies and their functional...
Surface binding autoantibodies and their functional effects in
dermatomyositis and polymyositis
Inaugural Dissertation
submitted to the
Faculty of Medicine
in partial fulfilment of the requirements
For the PhD-Degree
of the Faculties of Veterinary Medicine and Medicine
of the Justus-Liebig University Giessen, Germany
by
Pratibha Singh
from
Allahabad, India
Giessen 2010
From the Department of Neurology of the
Justus Liebig University Giessen
First Supervisor and Committee Member: Prof. Dr. Franz Blaes
Second Supervisor: Prof. Dr. Christiane Herden
Chairman of the examination panel: Prof. Dr. Wolfgang Kummer
Examiner: Prof. Dr. Stefan Arnhold
Date of the Doctoral Defense: 21st September, 2010.
Dedicated to my grand mother and my parents.................Dedicated to my grand mother and my parents.................Dedicated to my grand mother and my parents.................Dedicated to my grand mother and my parents.................
Declaration
I declare that the present thesis is my original work and that it has not been previously
presented in this or any other university for any degree. I have also abided by the
principles of good scientific conduct laid down in the charter of the Justus Liebig
University of Giessen in carrying out the investigations described in the dissertation.
Index
II
INDEX
INDEX ............................................................................................................................. II
ABBREVIATIONS.......................................................................................................... III
1. INTRODUCTION......................................................................................................... 1
1.1. Immune system............................................................................................................................... 1
1.2. Autoimmunity ................................................................................................................................. 4
1.3. Idiopathic inflammatory myopathies/ myositis ............................................................................. 7
1.3.1. Clinical features ......................................................................................................................... 7
1.3.2. Extramuscular manifestations .................................................................................................... 9
1.3.3. Epidemiology........................................................................................................................... 12
1.3.4. Etiology ................................................................................................................................... 14
1.3.5. Pathogenesis........................................................................................................................... 14
1.3.6. Treatment ................................................................................................................................ 21
2. AIM........................................................................................................................ 22
3. PATIENTS ............................................................................................................ 23
4. MATERIALS ......................................................................................................... 24
4.1. Chemicals and Solutions ............................................................................................................. 24
4.2. Consumables ............................................................................................................................... 25
4.3. Instruments ................................................................................................................................... 26
4.4. Molecular biology and biochemistry kits.................................................................................... 27
4.5. Buffers and Solutions................................................................................................................... 27
4.6. Media and solutions for cell culture ............................................................................................ 30
4.7. Cell lines........................................................................................................................................ 31
4.8. Antibodies..................................................................................................................................... 31
4.9. Primers.......................................................................................................................................... 32
4.10. Software ...................................................................................................................................... 32
5. METHODS ................................................................................................................ 33
5.1. Cell culture.................................................................................................................................... 33
5.2. Flow cytometry ............................................................................................................................. 33
5.2.1. Autoantibody detection by flow cytometry................................................................................ 33
5.2.2. MHC Class I detection ............................................................................................................. 34
5.3. RNA isolation, RT-PCR, and Real time PCR ................................................................................ 34
5.4. Cytotoxicity assay ........................................................................................................................ 35
5.5. Calcium imaging ........................................................................................................................... 35
Index
III
5.5. Protein extraction ......................................................................................................................... 36
5.5. ONE-DIMENSIONAL GEL ELECTROPHORESIS (1-DE).................................... 37
5.6. Two-dimensional gel electrophoresis (2-DE)......................................................................... 37
5.7. Protein identification by peptide mass fingerprinting........................................................... 38
5.8. Statistical Analysis ................................................................................................................. 38
6. RESULTS ............................................................................................................. 40
6.1. Presence of surface binding autoantobodies........................................................................ 40
6.2. Western blotting of autoantibodies with muscle cell extract................................................ 47
6.3. Identification of autoantigens in muscle cells....................................................................... 48
6.4. Cytotoxic effect of autoantibodies ......................................................................................... 50
6.5. Effect of autoantibodies on endothelial calcium flux............................................................ 53
6.6. Effects of statins on muscles cells ........................................................................................ 54
6.6.1. Statins enhance IFN-γ-induced MHC class I expression in TE671 but not in SKMC .......... 54
6.6.2. TAP and LMP expression ................................................................................................. 60
7. DISCUSSION........................................................................................................ 62
7.1. Surface binding autoantibodies................................................................................................... 62
7.2. Proteomic approach to identify autoantigens ............................................................................. 64
7.3. Functional effects of autoantibodies ........................................................................................... 66
7.4. Skeletal muscle cell MHC I expression in response to statin treatment .................................... 68
8. SUMMARY............................................................................................................ 74
9. ZUSAMMENFASSUNG........................................................................................ 75
10. REFERENCES...................................................................................................... 77
ACKNOWLEDGEMENTS............................................................................................. 96
PUBLICATIONS ........................................................................................................... 97
Abbreviations
III
Abbreviations
2D Two Dimensional
AECA anti endothelial cell antibodies
anti-ARS anti-aminoacyl tRNA synthetase antibodies
anti-Ku anti-Ku antibodies
anti-Mi-2 anti-Mi-2 antibodies
anti-PM-Scl anti-PM-Scl antibodies
anti-SAE anti-small ubiquitin-like modifier activating enzyme
anti-SRP anti-Signal-Recognition particle antibodies
anti-U1-RNP anti-U1-ribonucleoprotein antibodies
anti-U3-RNP anti-U3-ribonucleoprotein antibodies
AP Alkaline Phosphatase;
DM Dermatomyositis
E.Coli Escherichia coli
EC Endothelial cell
ELISA Enzyme Linked Immunosorbent Assay
FITC Fluorescinisothiocyanate
HCMEC Human chorionic micro-vascular endothelial cells
HDMEC human dermal micro-vascular endothelial cells
HUVEC Human umbilical vascular endothelial cells
IFN-γ Interferon gamma
IgG Immunoglobulin G
IIMs Idiopathic inflammatory myopathies
LEMS Lambert-Eaton Myasthenic syndrome
LMP2 / 7 Low molecular mass polypeptide 2 / 7
MAAs myositis-associated autoantibodies
MCTD Mixed connective tissue diseases
MHC class I Major histocompatibility complex class I
Abbreviations
IV
MSAs myositis-specific autoantibodies
mTOR mammalian target of rapamycin
OIM Other inflammatory myopathies
PBGD Porphobilinogen deaminase
PM Polymyositis
SEREX serological analysis of recombinant cDNA expression library
SERPA serological proteome analysis
SKMC Human primary skeletal muscle cells
SLE Systemic Lupus Erythematosus
TAP1 / 2 Transporter associated with antigen processing 1 / 2
TE671 Human rhabdomyosarcoma cells
WG Wegener’s granulomatosis
Introduction
1
1. Introduction
1.1. Immune system
The immune system is the body’s natural guardian against disease. It has evolved as a
way for us to defend ourselves from invading pathogenic organisms like bacteria,
parasites and viruses. It equals in complexity the intricacies of the brain and nervous
system, displays several remarkable characteristics which include distinction between
“self” and “non-self” and the ability to remember previous experiences and react
accordingly. Based on the type of response whether it is unspecific or specific, immune
system can be divided into innate and adaptive immune system respectively (Goldsby
et al. 2000; Janeway et al. 2001). These two systems are integrated and often interact
with each other (Hoebe et al. 2004).
Our first line of defence against any invasion from outside is the innate immune system,
which also includes a series of unspecific barriers that try to stop the invading
organisms. Our body surfaces are defended by epithelium, which provides a physical
barrier between the internal milieu and the external world that, contain pathogens.
Infections occur only when pathogen can colonize or pass through these barriers. The
low pH in the intestine and the presence of degrading enzymes also inhibit invading
pathogens. If a microorganism crosses the epithelial barrier and begins to replicate in
the tissues of host, in most cases it is immediately recognized by the mononuclear
phagocytes, or macrophages, that reside in these tissues (Fig. 1). While many cell types
are capable of endocytosis, only specialized cells like blood monocytes, tissue
macrophages and neutrophils are capable of phagocytosis. The next line of barrier also
includes the complement system, which consists of serum proteins that exist in from of
inactive proenzymes, which gets activated in response to different specific and non-
specific immunologic mechanisms. Once activated, they lead to direct damage to the
cell membrane of the pathogenic microorganisms or make them more prone to
phagocytic uptake by binding to the pathogens (opsonisation).
Introduction
2
Figure 1. Major events in the inflammatory response. A bacterial infection causes tissue damage
with release of various vasoactive and chemotactic factors. These factors induce increased blood
flow to the area, increased capillary permeability, and an influx of white blood cells, including
phagocytes and lymphocytes, from the blood into the tissues. The serum proteins contained in
the exudate have antibacterial properties, and the phagocytes begin to engulf the bacteria.
(Goldsby et al. 2000).
As is evident from the very name of it, “adaptive immune system”, it has the ability to
adapt to the changes in the threat from the pathogenic microorganisms. Four attributes
that characterize the adaptive immune system are: specificity, diversity, memory and
self / non-self recognition. The antigenic specificity of the immune system permits it to
distinguish subtle differences among the antigens. The immune system is capable of
generating tremendous diversity in generating its recognition molecules, allowing it to
recognize billions of unique structure on foreign antigens (Fig. 2). Some of the cell types
of the adaptive immune system are long lived and these cells contribute to the
immunologic memory that makes it possible for the immune system to remember
foreign molecules that it has experienced before. The next time the same foreign
Introduction
3
molecule is recognized again, the immune system can mount a specific response much
faster. Finally, the immune system normally responds only to foreign antigens,
indicating that it is capable of self/nonself recognition. It is intuitive to think that the only
important thing for the immune system is to defend the body from pathogenic
microorganisms, but not to attack the own body is of the same importance, as the
outcome of an inappropriate response to self molecules can be fatal.
Figure 2. Distinctive membrane molecules on lymphocytes. (a) B cells have about 105
molecules of membrane-bound antibody per cell. All the antibody molecules on a given B cell
have the same antigenic specificity and can interact directly with antigen. (b) T cells bearing
CD4 (CD4+ cells) recognize only extracellular antigen bound to class II MHC molecules. (c) T
cells bearing CD8 (CD8+ cells) recognize only intracellular antigen associated with class I MHC
molecules. In general, CD4+ cells act as helper cells and CD8+ cells act as cytotoxic cells. Both
types of T cells express about 105 identical molecules of the antigenbinding T-cell receptor
(TCR) per cell, all with the same antigenic specificity.(Goldsby et al. 2000).
An effective immune response involves two major groups of cells: lymphocytes and
antigen presenting cells. The lymphocytes can be of many different types and they are
all produced by haematopoiesis in the bone marrow. The major groups of lymphocytes
are T cells and B cells, and both these cell types are antigen specific in their mature
state. Lymphocytes leave the bone marrow, circulate in the blood and lymphatic system,
and reside in various lymphoid organs. Because they produce and display the antigen
binding cell-surface receptors, lymophocytes mediate the defining immunologic
attributes of specificity, diversity, memory and self / non-self recognition.
Introduction
4
B cells function as professional antigen presenting cells and they produce antibodies as
well as different cytokines. The antigen specific receptor of the T cells is called a T cell
receptor while the antigen specific receptors of B cells are membrane bound antibodies.
After binding of specific antigen to receptor antibodies, the antigen gets internalized and
processed into small peptides, which will then be expressed on the surface of B cells
with MHC class II. As soon as this MHC II-peptide is recognized by a T helper cell, it will
activate the B cell to start proliferating and differentiating into antibody producing
plasma cells or memory cells.
The antibodies produced by B cells are either secreted (plasma cells) or remain as
membrane bound receptors (memory cells). These molecules are antigen specific.
There are five different isotypes of antibodies with different functions. These antibodies
bind to antigens and result in opsonisation or complement activation (Kuby 1997).
1.2. Autoimmunity
The concept of autoimmunity was first predicted by Nobel Laureate Paul Ehrlich and he
described it as `horror autotoxicus´. We now understand that, while mechanisms of self-
tolerance normally protect an individual from potentially self-reactive lymphocytes, there
are failures. They result in an inappropriate response of the immune system against
self-components termed autoimmunity. Autoimmune diseases result from three
interacting components: genetic, environmental and regulatory (Ermann and Fathman
2001). Potential mechanisms of loss of self tolerance and potential induction of
autoimmunity could be:
1 Abnormal B, T cell signalling : Abnormal T cell activation and cell death
signalling underline the pathology of SLE (Kyttaris et al. 2005). Lupus B cells also
exhibit abnormal signalling through the B-cell receptor (BCR) (Jenks and Sanz
2009), mammalian target of rapamycin (mTOR) activation and enhanced Ca2+
flux (Fernandez et al. 2006).
2 Abnormal B, T cell apoptosis : impaired clearance of apoptotic cardiocytes, in
infants born to mothers with SLE or Sjogren’s syndrome has been linked to anti-
Introduction
5
SSA/Ro and –SSB/La antibodies in the pathogenesis of congenital heart block
(Clancy et al. 2006).
3 Abnormal autoantigen handling: abnormal clearance mechanisms can allow
the persistence of antigenic stimulation. Immune responses that specificall target
apoptotically modified form of lupus autoantigen have been identified in lupus
patients (Greidinger 2001).
4 Abnormal autoantigen modification: Although autoantigens targeted in
systemic autoimmune diseases share little in common in terms of structure,
subcellular distribution, or function in normal cells, these molecules are unified by
becoming clustered and concentrated in the surface blebs of apoptotic cells.
Furthermore, their structure is altered during some types of cell death to generate
structures not previously generated during development and homeostasis
(Rosen and Casciola-Rosen 2004).
5 Abnormal recognition of MHC class II: A striking characteristic of human
autoimmune diseases is the increased frequency of certain HLA class II alleles in
affected individuals. Since alleles positively associated with autoimmune
diseases share amino acid residues in the hypervariable HLA regions involved in
peptide binding, it is likely that disease associated class II molecules have the
capacity to bind the autoantigen and present it to T cells, thereby inducing and
maintaining the autoimmune disease (Adorini 1992).
6 Molecular mimicry: Although the triggering event in most autoimmune diseases
is unknown, an infectious cause has long been postulated to explain the
development of autoimmunity. Molecular mimicry is one mechanism by which
infectious agents (or other exogenous substances) may trigger an immune
response against autoantigens. According to this hypothesis a susceptible host
acquires an infection with an agent that has antigens that are immunologically
similar to the host antigens but differ sufficiently to induce an immune response
when presented to T cells. As a result, the tolerance to autoantigens breaks
down, and the pathogen-specific immune response that is generated cross-
Introduction
6
reacts with host structures to cause tissue damage and disease. In Guillain-Barré
syndrome antigenic epitopes are shared between Campylobactor glycoproteins
and structures of the myelin sheath (Albert and Inman 1999; Goodyear et al.
1999) .
5 Abnormal regulatory cells: The crucial role of regulatory cells in self-tolerance
and autoimmunity has been clearly established in numerous types of regulatory
cells, the majority of which are CD4+ T cells. Much focus has been placed on
thymically derived CD4+CD25+ regulatory T cells, given that the depletion of this
subset in murine models results in the spontaneous development of autoimmune
diseases (Asano et al. 1996).
6 Excessive polymorphism of autoantigens: Autoimmune diseases can be
either organ-specific or systemic. In an organ-specific autoimmune disease, the
immune response is directed to a target antigen unique to a single organ or
gland, so that the manifestations are largely limited to that organ. The cells of the
target organs may be damaged directly by humoral or cell-mediated effector
mechanisms. Alternatively, the antibodies may overstimulate or block the normal
function of the target organ. Few examples of this type of autoimmune disease
are Hashimoto’s thyroiditis, Goodpasture’s syndrome, Insulin-dependent
diabetes mellitus, which are mediated by direct cellular damage and others are
Graves’ disease, Myasthenia gravis, which are mediated by stimulating or
blocking autoantibodies (Besinger et al. 1983; Bach 1994; Drachman 1994). In
systemic autoimmune diseases, the response is directed towards a broad range
of target antigens and involves a number of organs and tissues. These diseases
reflect a general defect in immune regulation that results in hyperactive T cells
and B cells. Tissue damage is widespread, both from cell-mediated immune
responses and from direct cellular damage caused by autoantibodies or by
accumulation of immune complexes (Klinman 1989; Lin et al. 1991).
Myasthenia gravis (MG) usually presents in young adult or later adult life as muscle
weakness and excessive fatigue during repetitive movements. It most often involves the
Introduction
7
extraocular muscles of the eye with double vision and ptosis at onset, but usually
progresses to generalized weakness. Cholinesterase inhibitors, by prolonging the action
of ACh, tend to lead to clinical improvement. It is the prototype autoimmune disease
mediated by pathogenic antibodies. These autoantibodies are directed against various
nicotinic Acetylcholine receptor (AChR) on the neuromuscular junction or helper
proteins of the receptor such as the muscle-specific kinase MUSK (Vincent and
Newsom-Davis 1985; McConville et al. 2004). Interestingly, the anti-AChR antibodies do
not block the receptor directly, but they lead to a cross-linking of receptor molecules and
an internalisation of the receptor-antibody complexes (Elias et al. 1978; Appel et al.
1979).
1.3. Idiopathic inflammatory myopathies/ myositis
The idiopathic inflammatory myopathies comprise several diseases of which
polymyositis (PM), dermatomyositis (DM) and sporadic inclusion body myositis (IBM),
collectively called myositis are the most common. Apart from these three well-
characterised syndromes, some patients can have postinfectious or unclassified
myositis. The inflammatory myopathies are a heterogeneous group of subacute,
chronic, or acute acquired diseases of skeletal muscle (Dalakas and Hohlfeld 2003)
(Christopher-Stine and Plotz 2004). These disorders are characterized by a clinical
spectrum which includes muscle, skin and lung diseases, other associations including
cancer and specific autoantibodies (Bohan and Peter 1975 a; Bohan and Peter 1975 b;
Love et al. 1991; Targoff 2006; Targoff et al. 2006). Their etiology is not known but
genetic and environmental factors are believed to contribute to disease susceptibility
and to clinical phenotypes.
1.3.1. Clinical features
The most often used diagnostic or classification criteria for PM and DM were proposed
by Bohan and Peter in 1975 (Bohan and Peter 1975; Bohan and Peter 1975 a; Bohan
and Peter 1975 b). Later in a proposed revision of the Bohan and Peter criteria,
Magnetic resonance imaging to identify inflamed areas in muscle and autoantibodies
were also included. Although all classifications identify these disorders as part of a
Introduction
8
single disease spectrum, certain important features are used to separate subsets.
• Symmetrical proximal muscle weakness of limb-girdle muscles and anterior neck
flexors progressing over weeks to months, with or without disphagia or
respiratory muscle involvement.
• Elevation of serum skeletal muscle enzymes, particularly creatine phosphokinase
(CK) often aldolase, serum alanine aminotransferase (ALAT), aspartate
aminotransferase (ASAT) and lactate dehydrogenase (LD).
• Electromyographic changes (EMG) indicating muscular impairment i.e.
polyphasic short small motor neuron potentials, fibrillation, positive sharp waves,
increased insertional irritability, and repetitive high frequency discharges.
• Muscle biopsy evidence of Type I and II fibre phagocytosis, regeneration with
basophila, large vesicular sarcolemal nuclei and prominent nucleoli, atrophy in a
perifascicular distribution, variation in fibre size and inflammatory exudates, often
perivascular.
• Characteristic cutaneous manifestations of dermatomyositis (DM) including lilac
discoloration of eyelids (heliotrope) with periorbital edema, a scaly,
erythemathosis dermatitis over dorsum of the hands (especially the
metacarpophalengeal and proximal interphalangeal joints (Gottron’s sign)). This
type of distribution is considered to be virtually pathognomonic of DM.
Myositis (refers to all three subsets) patients within different subsets have distinct
clinical, histological and serological characteristics. Certain important features used to
separate these subsets include the distinctions between childhood and adult onset, PM
versus DM and the presence or absence of malignancy and other connective tissue
diseases. This suggests the involvement of different mechanisms in the development of
different subsets of myositis.
Most patients with PM or DM present with subacute or slowly progressive, usually
symmetric, proximal muscle weakness. These patients also suffer from decreased
Introduction
9
muscle endurance and fatigue, disabling them from daily activities.
DM is primarily characterized by muscular and cutaneous manifestations (Dalakas and
Hohlfeld 2003; Briani et al. 2006; Callen and Wortmann 2006). The onset of DM may be
acute or insidious and distal muscles being involved very late. The extensor muscles of
the neck may be involved, causing difficulty in holding up the head. Myositis patients
with severe muscle weakness can potentially end up wheelchair-bound and requiring
assisted ventilation. The most common clinical signs are reduction of the muscular
strength in the proximal muscles, contractures and, late in the course of the disease,
muscular atrophy.
1.3.2. Extramuscular manifestations
1.3.2.1. Cutaneous manifestations: Dermatomyositis is identified by a characteristic
rash accompanying or, preceding muscle weakness. The most common and peculiar
skin manifestations are: (1) violaceous erythematous papules which may be observed
on the extensor surface of metacarpophalangeal, proximal and distal interphalangeal
joints (Gottron’s sign) (Fig. 3 A), while Gottron’s papules are found covering other bony
prominences such as elbows, knees etc; (2) heliotrope rash, a purplish erythema, with
or without oedema, in a symmetrical distribution involving periorbital skin (Fig. 3 B).
Several other cutaneous features, characteristic of the disease are malar erythema,
periungual teleangiectasia with or without dystrophic cuticles, vasculitic skin
manifestations consisting of subcutaneous nodules, erythema, periungual infarctions,
and digital ulcers, calcification in the subcutaneous tissues leading to subcutaneous
painful hard nodules, and Raynaud’s phenomenon which is more common in patients
with idiopathic DM and in DM associated with connective tissue diseases.
Introduction
10
A. B.
Figure 3. Rash in dermatomyositis. (A) Gottron’s rash (circled); (B) Heliotrop rash (circled).
In children, DM resembles the adult disease, except for more frequent subcutaneous
calcifications, vasculitic skin changes, and extramuscular manifestations.
DM can be associated with cancer or may overlap with systemic sclerosis and mixed
connective-tissue disease. Patients with inflammatory myopathies have higher risk of
malignancy than the normal population. In DM, it has been reported to occur in
approximately 30% of cases with a higher occurrence in men and in old age (Dalakas
and Hohlfeld 2003; Briani et al. 2006; Callen and Wortmann 2006).
1.3.2.2. Pulmonary disease: Pulmonary complications constitute important clinical
manifestations of PM or DM. The lungs may be involved either primarily or as a
complication of muscle weakness. The reported prevalence of lung involvement in PM
and DM varies from 5 to 65 %, (Hepper et al. 1964; Marie et al. 1998; Douglas et al.
2001; Fathi et al. 2004) depending on whether clinical, radiological, functional or
pathological criteria have been used. The most common pulmonary disorder in PM and
DM is interstitial lung disease (ILD), not always related to muscle symptoms and is one
of the hallmarks of anti-synthetase syndrome. ILD can appear before, at the same time
or after the onset of skin or muscle symptoms. The presence of ILD in patients with
Introduction
11
myositis affects the prognosis, with increased morbidity and mortality, and this often
also has an influence on the choice of immunosuppressive treatment.
1.3.2.3. Heart involvement: The clinical cardiac manifestations most frequently
reported in IIMs, myositis, are congestive heart failure, conduction abnormalities and
coronary artery disease. The frequency of heart involvement in myositis varies between
6% and 75% depending on patient selection, definition of heart involvement, whether
clinical manifest or subclinical cardiac involvement is considered, and methods used to
detect cardiac involvement (Bohan et al. 1977; Gottdiener et al. 1978; Taylor et al.
1993). Also children with juvenile DM may develop cardiac involvement although the
frequency seems to be low. ECG abnormalities observed in PM and DM patients
include atrial and ventricular arrhythmias, bundle branch block, A-V blocks, high- grade
heart block, prolongation of PR-intervals, ventricular premature beats, left atrial
abnormality, abnormal Q-waves, as well as non-specific ST-T wave changes.
Though clinically significant cardiac involvement is infrequent in PM and DM, but
cardiovascular manifestations constitute a major cause of death (Bohan et al. 1977 ;
Hochberg et al. 1986). Based on available data it can be suggested that inflammation of
the heart muscle could be important in the pathophysiologic mechanisms which could
lead to myocarditis and congestive heart failure or conduction disturbances in patients
with myositis.
1.3.2.4. Other organ involvement: Other organs that are frequently involved in
myositis are the pharyngeal and gastrointestinal muscles. The most common GI
symptom is dysphagia and disordered esophageal motility.
1.3.2.5. Malignancy: Although all the inflammatory myopathies can have a chance
association with malignant disease, especially in older age-groups, the frequency of
cancer is definitely increased in DM (Sigurgeirsson et al. 1992; Buchbinder et al. 2001).
The most common cancers are those of the ovaries, gastrointestinal tract, lung, and
breast and non-Hodgkin lymphomas (Hill et al. 2001). The increased risk is both at the
time of DM diagnosis but also after more than 10 years duration of disease (Callen
2002). There have been some recent reports linking polymyositis also with malignancy
Introduction
12
of one or the other type. These reports include: paraneoplastic polymyositis associated
with; squamous cell carcinoma of the lung (Gabrilovich et al. 2006); with transitional cell
carcinoma of the bladder (Bouropoulos et al. 1997); thymic carcinoma (Inoue et al.
2009) and a renal carcinoma (Wurzer et al. 1993).
1.3.2.6. Overlap syndromes: PM and DM are seen in association with various
autoimmune and connective tissue diseases (Table 1). The term overlap syndrome
indicates that certain clinical signs are shared by both disorders. Myositis-overlap
syndromes are characterized by a heterogeneous group of clinical syndromes of which
many are closely linked with specific autoantibodies. It is only DM, and not PM, that truly
overlaps and that too only with systemic sclerosis and mixed connective-tissue
diseases. In a recent report DM has been shown to overlap with lupus (Dayal and
Isenberg 2002).
Table 1. Conditions and factors associated with inflammatory myopathies (Dalakas and Hohlfeld
2003).
1.3.3. Epidemiology
The IIMs are rare disorders and the reports of incidence and prevalence are limited. In
PM/DM several different classification systems have been proposed. Although all
classifications identify these disorders as part of a single disease spectrum, certain
important features are used to separate subsets. The different incidences in different
population based studies, might be due to varying inclusion criteria as well as varying
case retrieval strategies used in these studies, which makes comparisons difficult, but
Introduction
13
ethnic or geographical differences can not be excluded (Benbassat et al. 1980; Oddis et
al. 1990; Weitoft 1997).
1.3.3.1. Incidence by Age, Race and Sex: Although inflammatory myopathy can occur
at any age, the observed pattern of incidence includes childhood and adult peak and a
paucity of patients with onset in the adolescent and young adult years. The incidence
sex ratio is: 2.5:1 female to male (Benbassat et al. 1980; Oddis et al. 1990; Leff et al.
1991; Symmons et al. 1995; Vegosen et al. 2007). This ratio is lower (nearly 1:1) in
childhood disease and with associated malignancy, but is very high 10:1 when there is
an associated connective tissue disease. PM/DM has a 3-4:1 Black to White incidence
ratio.
1.3.3.2. Environmental factors: The etiology of myositis is most likely interplay
between genetic susceptibility and exposure to certain environmental factors. Disease
onset is more frequent in the winter and spring months, especially in childhood cases,
consistent with precipitation by viral and bacterial infections. Seasonal patterns in the
onset of myositis characterized by disease-specific autoantibodies such as anti-Jo1 and
anti-signal recognition particle (SRP) has been reported, indicating a common
environmental factor contributing to the disease (Leff et al. 1991; Vegosen et al. 2007).
In one study, PM/DM patients reported excessive physical exercise, antedating illness
significantly more frequently than controls, but this association has not been confirmed.
1.3.3.3. Genetic susceptibility: Myositis is associated with certain HLA-DR genotypes
(Arnett et al. 1996; Shamim et al. 2000; Badrising et al. 2004; O'Hanlon et al. 2006).
White children with DM and adults with PM have an increased frequency of HLA-
B8/DR3, and HLA-B14 and B40 have been observed more commonly in adults with DM
coexisting with another connective tissue disease. HLA is considerably more closely
linked to several recently identified serum autoantibodies which have been found to
define clinically homogenous patient groups. Anti-Jo1 antibody patients have a
significantly increased frequency of HLA-DRw52 compared with control person, and
those with anti-PM-Scl, nearly all possess HLA-DR3 or DRw52.
Introduction
14
1.3.3.4. Viral infections: Many patients with myositis have reported that their clinical
symptoms have appeared and persisted after a cold or flu episode. Infection with
different viruses such as coxacievirus, adenovirus, parvovirus B19 and the retrovirus
human immunodeficiency virus (HIV) has been associated with myositis (Travers et al.
1977; Dalakas et al. 1986; Harland et al. 1991; Jongen et al. 1994; Chevrel et al. 2000;
Crowson et al. 2000; Douche-Aourik et al. 2003; Dalakas et al. 2007). Potential
mechanisms through which an infection can lead to an autoimmune disease process
include molecular mimicry, epitope spreading, bystander activation, polyclonal activation
and viral superantigen activation (Wucherpfennig 2001).
1.3.4. Etiology
As the name idiopathic inflammatory myopathies, the causes of the PM or DM are
unknown. However, infectious agents, drugs, toxins, genetic factors all have claimed for
attention as etiologic agents in IIMs. The rarity of myositis has precluded concordance
studies in twins, but reports of multicase families support a familial predisposition
(Shamim et al. 2000). According to a recent study different immunogenetic profiles
influence both clinical phenotype and the pattern of circulating myositis-specific
autoantibodies (Chinoy et al. 2006).
A recent work provides evidence that seasonal birth distributions of Hispanic juvenile
IIM patients, juvenile IIM patients with the p155 autoantibody, and juvenile patients with
certain HLA alleles differ from the birth distributions of patients in other subgroups or
from the birth distribution of a population of individuals not known to have an
autoimmune disease. It also suggests that early environmental influences have a
greater influence on childhood-onset myositis than on adult-onset myositis (Vegosen et
al. 2007).
1.3.5. Pathogenesis
1.3.5.1. Immune mechanisms: The causes and pathogenesis of myositis remains
unclear but immune mechanisms are strongly implicated. The autoimmune origin of
these diseases is supported by their association with other autoimmune disorders,
autoantibodies (Targoff 2002), and histocompatibility genes; the evidence of T-cell-
Introduction
15
mediated myocytotoxicity or complement-mediated microangiopathy, and their response
to immunotherapy. Investigations of skeletal muscle tissue suggest that both the
skeletal muscle fibres and the microvasculature could serve as targets of the immune
response through involvement of mononuclear inflammatory cells and their mediators
(Hohlfeld and Engel 1994; Targoff 2000; Dalakas 2002 a; Dalakas and Hohlfeld 2003).
1.3.5.2. Inflammatory infiltrates: Based on major patterns of inflammatory infiltrates in
skeletal muscle tissue in myositis, these diseases can be distinguished: (a) Endomysial
infiltrates with primarily CD8+ T cells surrounding and invading muscle fibers and with
macrophages, less common CD4+ T cells, mainly observed in patients with PM and
IBM; (b) Perivascular infiltrates predominantly composed of CD4+T cells, macrophages
and occasionally B cells are mainly detected in DM patients (Fig. 4 A and B) (Arahata
and Engel 1984; Engel and Arahata 1984). Though these diseases share three
dominant histological features that are ultimately responsible for the clinical signs of
muscle weakeness: inflammation, fibrosis and loss of muscle fibers, yet they are
clinically and immunopathologically distinct (Engel and Arahata 1984; Hohlfeld and
Engel 1994; Dalakas 1995).
Figure 4. (A) H & E staining of a muscle from Dermatomyositis patient showing perivascular
infiltration. (B) H & E staining of a muscle from Polymyositis patient showing endomysial
infiltration.
Introduction
16
1.3.5.3. Immune cells in myositis: T cells are frequently present in the muscle tissue
in all subsets of myositis but with large individual variations. Electron microscopy
studies of inflamed muscle tissue from polymyositis patients suggested that CD8+T
cells are cytotoxic to muscle fibres (Arahata and Engel 1986). These CD8+ as well as
CD4+ muscle-infiltrating T cells have been shown to be perforin positive (Goebels et al.
1996), suggesting a possible T cell-muscle cell interaction. In patients with PM and IBM,
but not in DM, only certain T cells of specific TCRα and TCRβ families are recruited to
the muscle from the circulation (Mantegazza et al. 1993; O'Hanlon et al. 1994a; Bender
et al. 1995). Cloning and sequencing of the amplified endomysial or autoinvasive TCR-
gene families have demonstrated a restricted use of the Jβ gene with a conserved
amino acid sequence in the CDR3 region, indicating that CD8+ cells are specifically
selected and clonally expanded in situ by muscle specific autoantigens (Salajegheh et
al. 2007). A cytotoxic effect of T cells is still a subject of controversy since no muscle-
specific antigens have been identified and since an expression of the co-stimulatory
molecules CD80/86, normally required for functional interaction, has not been detected
in inflamed muscle fibres (Yamada et al. 2001).
Though inflammatory cell infiltrates in muscle tissue, often decrease after conventional
immunosuppressive treatment, yet in some cases the inflammatory cells may persist
(Bunch et al. 1980; Lundberg and Chung 2000; Korotkova et al. 2008).
1.3.5.4. Immunological molecules: In the skeletal muscle tissue of healthy individuals
only the presence of occasional mononuclear cells or expression of immunological
molecules can be observed (Malm et al. 2000; Dorph et al. 2006). Inflammatory cells,
adhesion molecules, pro-inflammatory cytokines and chemokines detected by
immunohistochemistry are usually over-expressed in muscle tissue of myositis patients
compared to in healthy individuals, leading to the belief of their involvement in the
pathogenesis of myositis. Complement activation leading to the formation and
deposition of membranolytic attack complex on the endomysial microvasculature,
resulting in capillary necrosis, inflammation, ischemia and perifscicular atrophy has
been seen in DM (Hohlfeld and Engel 1994; Dalakas 2002 b; Dalakas and Hohlfeld
2003) while in polymyositis an antigen-directed and MHC class I restricted CD8 T cel
Introduction
17
mediated cytotoxicity has been implicated. The above is supported by following reports:
the cytotoxicity of endomysial T cells to autologous myotubes (Hohlfeld and Engel
1994); the clonal expansion of autoinvasive T cells and the restricted usage of T cell
receptor gene families (Mantegazza et al. 1993; O'Hanlon et al. 1994b; Bender et al.
1995; Amemiya et al. 2000; Benveniste et al. 2001; Nishio et al. 2001); the upregulation
of co-stimulatory molecules (Behrens et al. 1998; Murata and Dalakas 1999); and the
release of perforin granules by autoinvasive CD8 cells to lyse muscle fibres (Goebels et
al. 1996). Upregulated chemokines, cytokines and adhesion molecules enhance the
transmigration of T cells from the circulation to the muscle (Choi and Dalakas 2000;
Figarella-Branger et al. 2003). What triggers complement activation in DM or T cell
activation in PM still remains unclear.
1.3.5.5. Humoral immunity: Presence of B cells in DM and to a lesser extent in PM
and IBM patients support the involvement of humoral adaptive immunity in these
diseases (Arahata and Engel 1984; Greenberg et al. 2005; Bradshaw et al. 2007). It
appears that the disease is driven, at least partly, by a loss of self tolerance with the
production of autoantibodies. Up to 80% of patients with PM or DM, but less commonly
in patients with IBM, have autoantibodies (Levine 2005; Noss et al. 2006). Evidently,
plasma cells detected in muscle tissues of PM and IBM patients had undergone
oligoclonal expansion, and affinity maturation as well as isotype switching had occurred
in individual cells, suggesting that antigen drives a B cell antigen-specific response in
muscle tissue of myositis patients (Bradshaw et al. 2007).
The most common autoantibodies are antinuclear autoantibodies. Some of the
autoantibodies are often found in other inflammatory connective tissue diseases (for eg.
Anti-PMScl, anti-SSA, and anti-SSB) and are called myositis-associated autoantibodies.
Other autoantibodies, called myositis specific autoantibodies, are more specific for
myositis, although they might not be found exclusively in myositis but occasionally in
other patients. Recent investigations have demonstrated that about 20% to 30% of
myositis patients have autoantibodies seen mainly in patients with myositis. Anti-Jo-1
(antihistidyl-tRNA synthetase) is the most prevalent MSA (myositis specific antibodies)
and the most common of the eight anti-aminoacyl-tRNA synthetase autoantibodies
Introduction
18
(anti-ARSs) currently described (Nishikai and Reichlin 1980; Mimori et al. 2007;
Gunawardena et al. 2009a). These autoantibodies target cytoplasmic enzymes that
catalyze the binding of specific amino acids to their cognate tRNA and define the anti-
synthetase syndrome (ASS) (Targoff and Reichlin 1985). Autoantibodies to Mi-2 are
detected in patients with hallmark dermatomyositis features. Mi-2 is a nuclear helicase
protein that forms part of the nucleosome remodelling deacetylase (NuRD) complex
playing a role in gene transcription (Wang and Zhang 2001). In recent years, a number
of novel MSAs have also been described. Sato et al. (Sato et al. 2005) identified an
autoantibody in Japanese patients with clinically amyopathic dermatomyositis (CADM)
and rapidly progressive lung disease. This autoantibody (known as anti-CADM140)
targets a cytoplasmic 140-kDa protein melanoma-differentiation associated gene 5
(MDA5). This autoantigen is involved in the innate immune defence against viral
infections through the detection of viral dsDNA (Takeuchi and Akira 2008). In addition,
anti-p155/140 autoantibodies have been reported in adult and juvenile dermatomyositis
(Targoff et al. 2006; Kaji et al. 2007; Chinoy et al. 2007 a). Targoff et al. identified
p155/140 as transcriptional intermediary factor 1-gamma (TIF1-g), a nuclear protein
involved in cellular differentiation. The most striking feature of anti-p155/140
autoantibodies is the association with malignancy in adults. Gunawardena et al. have
described autoantibodies to a p140 kDa target in juvenile dermatomyositis
(Gunawardena et al. 2009b). The p140 kDa target has recently been identified as
nuclear matrix protein NXP-2, involved in nuclear transcription. Finally, Betteridge et al.
have described anti-SAE (small ubiquitin-like modifier activating enzyme)
autoantibodies in adult dermatomyositis patients (Betteridge et al. 2007; Betteridge et
al. 2009 a). The target autoantigen, small ubiquitin like modifier-activating enzyme is a
protein involved in posttranslational modification that is located in both the nucleus and
cytoplasm of cells (Dohmen 2004).
1.3.5.6. Major histocompatibility complex (MHC) class I antigen: The Major
Histocompatibility Complex (MHC) is a set of molecules displayed on cell surfaces that
are responsible for lymphocyte recognition and "antigen presentation". The MHC
molecules control the immune response through recognition of "self" and "non-self". The
Class I and Class II MHC molecules belong to a group of molecules known as the
Introduction
19
Immunoglobulin Supergene Family, which includes immunoglobulins, T-cell receptors,
CD4, CD8, and others. Class I MHC molecules bind peptides and present them to
CD8+T cells. In general these peptides are derived from enoodgenous intracellular
proteins that are digested in the cytosol. The peptides are then transported from the
cytosol into the cisternae of the endoplasmic reticulum, where they interact with class I
MHC molecules. This process is know as the cytosolic or endogenous processing
pathway (Fig. 5).
Muscle fibres of most myositis patients, in contrast to those of healthy individuals,
express MHC class I antigen. The over-expression of MHC class I molecules is an early
event in many autoimmune diseases, since it is a prerequisite for the cytolytic action of
cytotoxic T lymphocytes. MHC class I molecules by themselves can have a deleterious
effect on cell types that do not constitutively express these molecules (Nagaraju 2005).
Normal human skeletal myoblasts constitutively express low levels of MHC class I
molecules under cell culture conditions (Hohlfeld and Engel 1991; Nagaraju et al. 1998).
Muscle fibres of healthy individuals do not express detectable levels of MHC class I
antigens, although these fibres have been shown to express MHC class I in several
autoimmune muscle diseases (Appleyard et al. 1985; Emslie-Smith et al. 1989). A
transgenic mouse model that constitutively over-expresses MHC class I in skeletal
muscle fibres develops clinical, biochemical, histological and immunological features
similar to those of human myositis (Nagaraju et al. 2000). Some investigators advocate
characterize DM patients histologically by MHC class I antigen expression
predominantly located to perifascicular muscle.
The explanation for the MHC class I expression on muscle fibres in human myositis is
unknown. An endoplasmic reticulum stress response is suggested to be a link between
MHC class I up-regulation and muscle damage and dysfunction in PM and DM. Over-
expression of MHC class I induced an ER-stress response in mouse model, by up-
regulating nuclear factor (NF)-kB, which is strongly activated in myositis and can
suppress myoblast differentiation and induce pro-inflammatory cytokines causing
muscle damage.
Introduction
20
Figure 5. Cytosolic and nuclear proteins are degraded by the proteasome into peptides. The
transporter for antigen processing (TAP) then translocates peptides into the lumen of the
endoplasmic reticulum (ER) while consuming ATP. MHC class I heterodimers wait in the ER
for the third subunit, a peptide. Peptide binding is required for correct folding of MHC class I
molecules and release from the ER and transport to the plasma membrane, where the peptide is
presented to the immune system. TCR, T-cell receptor (Kuby 1997).
1.3.5.7. Enhanced autoantigen expression: Casciola-Rosen et al demonstrated
enhanced expression of both Mi-2 and Jo-1 in myositis muscle compared with normal
muscle, and this was primarily observed in regenerating muscle rather than in mature
myotubes (Casciola-Rosen et al. 2005). They also showed that myositis autoantigen
expression is also markedly increased in several cancers known to be associated with
autoimmune myositis, but not in their related normal tissues, showing the similarity
between tomor cells and undifferentiated myoblasts. Work from Casciola-Rosen et al.
Introduction
21
and Zampieri et al supports the hypothesis that the presence of candidate myositis
autoantigens during reparative myogenesis and in cancer-associated myositis, an
autoimmune response directed against cancer, cross reacting to regenerating muscle
cells can drive induction and propagation of the autoimmune response (Casciola-Rosen
et al. 2005; Zampieri et al. 2008).
1.3.6. Treatment
The general recommended treatment of PM and DM is based on immunosuppressive
agents, starting with high dosage of glucocorticosteroids often in combination with
methotrexate, azathioprine (Oddis 1994; Adams and Plotz 1995; Mastaglia et al. 1997).
Glucocorticosteroids can reduce the inflammatory infiltrates in muscle tissue and
thereby decrease the expression of some pro-inflammatory molecules and adhesion
molecules (Lundberg and Chung 2000).
In a placebo-controlled trial high-dose IVIg was beneficial in treatment resistant DM
patients, both regarding muscle function and muscle histopathology (Dalakas et al.
1993). Although the clinical effects were accompanied by decreased ICAM-1 and MHC
class I expression in repeat muscle biopsies, the number of patients that were subject to
repeat biopsy was small. In case of PM patients treated with IVIg, only the clinical
reports are available to date (Cherin et al. 1990; Cherin et al. 1991; Cherin et al. 2002;
Danieli et al. 2002). So before plunging to start treating myositis patients with IVIg,
further investigation of the underlying molecular mechanisms is necessary.
There are few studies reporting varying effects of treatment with tumor necrosis factor
(TNF) blockers in patients with PM or DM. TNF blockade has been inconsistent and
even worsened disease in some reported cases (Hengstman et al. 2003; Hengstman et
al. 2004), again underscoring the need to understand the molecular mechanisms
involved in these diseases.
Aim
22
2. Aim
Since inflammatory histopathology could be found in the muscles of IIM patients and
steroids or other immunosuppressantrs are helpful in DM/PM, an autoimmune etiology
was proposed in these diseases. Aim of the study was to identify and characterize the
muscle specific autoantigens in IIMs, using different autoantibody detection methods
and protein biochemistry approach, such as 2-DE and mass spectrometry. The project
aimed at answering following questions:
First, do the patients have muscle-specific or endothelial cell specific autoantibodies? If
yes, which are the underlying autoantigens?
Second, do the autoantibodies from myositis patients have any functional effects?
Thirdly, do the statins, reported to stimulate expression of MHC class I on muscle cells
leading to the development of myopathy, affect expression of MHC class I , TAP and
LMP ( involved in class I antigen presentation) in vitro on muscle cells?
Patients
23
3. Patients
Serum was obtained from 41 patients with inflammatory muscle diseases (mean age
50.3 ± 17.8 years; 24 female / 17 male) after informed consent and approval of the local
ethical committee. 11 patients had polymyositis and 15 patients had dermatomyositis
according to the criteria of Bohan and Peter (Bohan and Peter 1975; Bohan and Peter
1975 a; Bohan and Peter 1975 b). The other myositis patients (n=15) had unclassified
or postinfectious myositis. The epidemiological data of the groups are given in table 2.
The mean duration of myositis at the time the serum sample was obtained was 1.1 ± 0.7
years. Sera of 26 healthy people (HC) served as controls (mean age 51.5 ± 18.2 years,
15f / 11m).
Diagnosis n Mean age ±±±± StDev Female / Male
Dermatomyositis 15 42.4 ±±±± 19.0 12f / 3m
Polymyositis 11 63.1 ±±±± 12.1 6f / 5m
Unclassified
myositis 15 49.2 ±±±± 14.9 6f / 9m
Table 2. Patient data of myositis patients.
Materials
24
4. Materials
4.1. Chemicals and Solutions
Acetic Acid 100% Merck Agarose multipurpose Bioline GmbH Ammoniumpersulfat APS Roth Antioxidant Invitrogen 5-bromo 4-chloro 3-indolyl phosphate / nitro-blue tetrazolium chloride (BCIP/NBT)-Blue Liquid Substrate
Sigma Aldrich
BDH crystalline Trypsin (bovine pancreas) Sigma Aldrich Bovine Serum Albumine, Fraction V (BSA) Sigma Aldrich Bromphenolblue Neolab β-Mercaptoethanol
Fluka
CelLyticTM M Cell Lysis Reagent Sigma Deoxiribunuclease 1, Type 4 bovine pancreas Sigma Aldrich Destilled Water „Aqua ad injectabilia“ Braun D(+)Glucose Gibco Dimethylsulfoxide (DMSO) Roth Ethylendiamintetracetic acid (EDTA) 0,5M Roth Ethanol 100 % Sigma Aldrich Ethanol for Molecularbiology 100% Merck Ethidiumbromid Merck Ficoll-PaqueTM Plus Amersham 5-Fluoro-2´-deoxyuridine Merck Glycerol Roth Glycin Merck H-EBSS Gibco Hepes Sigma Interferon γ (IFN-γ) Provitro IgG-Standard Sigma Isopropanol Merck Potassiumchlorid (KCl) Merck Potassiumdihydrogenphosphat (KH2PO4) Merck 4x SDS sample Buffer (for WB) Invitrogen
Methanol Merck Magnesiumsulphate (MgSO4) Sigma
Sodiumchloride (NaCl) Roth
Sodiumhydrogencarbonate (NaHCO3) Merck Disodiumhydrogenphosphate (Na2HPO4) Merck
Sodiumdihydrogenphoaphate (NaH2PO4) Merck
Sodiumazide (NaN3) Merck
Non-fat dry milk, blotting grade Bio-Rad
Materials
25
NuPage SDS MOPS Running Buffer Invitrogen PageRuler™ Prestained Protein Ladder Fermentas
Paraformaldehyde (PFA) Sigma Aldrich
Phosphate Buffered Saline (PBS) 10x (for cellcultur!)
Gibco
Poly-L-lysin-hydrobromide Sigma
Ponceau S Sigma / Roth
Protease Inhibitor Cocktail Sigma Protein G-Sepharose GE Healthcare Bio-Science AB Quick Load 1kb DNA Ladder Biolabs
RNAse free Water
Rotiphorese Gel 30 = 30 % Acrylamide-Mix Applichem
Sample reducing Agent (10x) Roth
Saponin Invitrogen
Sodiumdodecylsulfate (SDS) Sigma
Tris-Acetat-EDTA Buffer (TAE) 10x Neolab Trichlor-Acetic acid (20%) Roth
Trishydroxymethylaminomethan (Tris) Roth
Tris-HCl Sigma
Trypanblue USB Trypsin (2,5g/l) Roth
5% Trypsin-EDTA (10x) Gibco
Trypsin inhibitor Type I-S: from Soybean Gibco Trypsin Type XII-S Sigma Aldrich
Tween20 Sigma Aldrich
4.2. Consumables
Cellstar® 6 Well Cell Culture Plate Greiner bio-one Cellstar® Plastikpipettes (5 ml, 10 ml) Greiner bio-one Cellstar® U-shape with Lid, TC-Plate, 96 well, sterile Greiner Bio-one Cellstar® 75 cm2 Cell cultur flasks Greiner Bio-one Cryobox 136x136x130 mm Ratiolab GmbH Cryo TubeTM vials (1,8 ml; 4,5 ml) Nunc Disposable scalpel, sterile Feather safity razorco 2D-well Gradient gel NuPage 4-12% (1,0 mm thick) Invitrogen Disposable cuvettes Ratiolab FACS-Tubes 0,5 ml 38x6,5 mm PS Sarstedt Falcon 5ml Polystyrene Round-Bottom Tube Becton Dickinson Falcon® Plastic pipettes 25 ml Becton Dickinson Labware Falcon tubes (15 und 50 ml) Becton Dickinson Gel documentation Thermal Image System FTI-500 Fuji Film Tissue culture dishes steril 35,0 / 10 mm Greiner bio-one Glaswares (different sorts) Fisherbrand; IDL; Schott&Gen;
Materials
26
Simax Kodan® Tinktur Forte (alcoholic skin disinfectant) Schülke & Mayr LightCycler® Capillaries [20 µl] (for Real time-PCR) Roche Minisart single use filter (0,2 µm, 0,45 µm) Biotech Neubauer improved Brand Nitra-Tex® powder free Ansell Nitrocellulose membrane Biometra NobaGlove® –Latex powder free NOBA GmbH NunclonTM surface 96-Well plates with flat bottom NuncTM Parafilm American National Can Glas Pasteur pipettes 150 mm Brand Pipette tipps (10µl, 100µl, 1000µl) Sarstedt PP-PCR-Tubes 0,2ml thin walled Greiner bio-one Grid inserts for Cryobox Ratiolab GmbH Reaction tubes 1,5 ml Sarstedt Safety-Multifly®–Set, sterile, pyrogenfree (Cannulae)
Sarstedt
Servapor® dialysis tubing (6mm, 25mm) Serva Electrophoresis GmbH S-Monovette® 7,5 ml Z (Serum-Tubes) Sarstedt Sterile Pipette tips with filter Nerbe Plus UV-spectroscopic cuvettes Bio-Rad Whatmann-Filterpaper 3 mm A. Hartenstein Cell scrapper Greiner bio-one
4.3. Instruments
Liquid Nitrogen tank Arpege 75 Blotapparatur Invitrogen ClasII Type A/B3 (Sterilbank) Nuaire Biological Safety
Cabinets Easia shaker Medgenix diagnostics FACSCalibur Becoton Dickinson Fluorescence microscope DM RB Leitz Gel documentation Image Masters VDS Pharmacia Biotech Gel electrophoresis chamber Peqlab Gel trays und Gel combs Peqlab Heating block / Thermoshaker Peqlab HiTrapTM Protein G HP (1ml und 5ml Protein G columns)
Amersham Biosciences
Inverse Light microscope MBL 3100 A.Krüss Optronic Refrigerators and Freezers Bosch, Liebherr, Nuaire, Santo,
Premium Light Cycler 1.5 Roche Diagnostics LightCycler centrifuge adapter Roche Diagnostics
Materials
27
Magnetic mixer IKA® Werke Microwell SHARP Electronics Multiscan Ex (ELISA-Reader) Thermo Electron Corporation NalgeneTM Cryo 1 °C Freezing Container Nalgene® NOVEX Xcell surelock Blotting chamber Invitrogen PC-System, Printer Hewlett Packard pH-Meter Schott Geräte Pipettes (different volumes) Gilson, Eppendorf Pipette boy Integra Biosciences ProSpec (Nephelometer) Dade Behring Pump P-1 (Pump for IgG purification) Pharmacia Biotech Rotamax 120 (Shaker) Heidolph Swivel platform Peqlab SmartSpecTM Plus Spectrophotometer Bio-Rad Sonopuls HD 2070 (Ultra sonicator) Bandelin Elektronik Power pack Peqlab Steri-Cult 200 Incubator for cell culture Labotec GmbH Sterile bench Köttermann Thermocycler Biometra Table top centrifuge EBA 20 Hettich Table top centrifuge micro 120 Hettich Universal 32 R (centrifuge) Hettich Vortex Minishaker IKA® Werke Vortexer Vortex-Genie2 Scientific Industries Weighing balance Sartorius AG Waterbath Memmert Centrifuge Typ 2-6 Sigma Centrifuge Universal 32 R (cell culture) Hettich
4.4. Molecular biology and biochemistry kits
Cytotoxicity Detection Kit (LDH) Roche Applied Science Quanti FastTM SYBR Green PCR Kit Qiagen RevertAidTM First Strand cDNA Synthesis Kit Fermentas
4.5. Buffers and Solutions
DNA-loading buffer (10x): 250 mg 33 ml 60 ml 7 ml
Bromphenolblue Tris (150 mM, pH 7.6) Glycerol H2O
Materials
28
Ethidium bromide staining solution 2.5 mg
1 L
Ethidiumbromide 1x TAE
FACS buffer 500 ml 5 ml 5 ml
1x PBS 10 % NaN3
Fetal calf serum (FCS)
Glycine buffer 3.75 g In 500 ml
Glycine dH2O pH 9.0
IgG-Elution buffer 0.75 g 100 ml
Glycine dH2O pH 2.7 (= 0.1 M)
Blocking buffer 0.4 g 10 ml
Non fat dry milk PBS Tween
1 % Paraformaldehyde (PFA) 1 g 100 ml
PFA 1x PBS
PBS (10x)
80 g 2 g
14.4 g 2.4 g
Dissolve in 1 L
NaCl KCl Na2HPO4
KH2PO4
dH2O
1x PBS 900 ml 100 ml
dH2O PBS (10x)
PBS Tween 1 L 500 µl
1x PBS Tween 20
Permeabilisation buffer 500 ml 0.5 g
FACS buffer Saponin
Poly-L-Lysin 60 ml 3 mg
dH2O Poly-L-Lysin
Ponceau S-solution 0.25 g 15 ml Make up
100 ml
Ponceau S Trichloracetate (TCA) with H2O (protect from light)
Materials
29
Protein (SDS)-loading-Buffer 4x
25 ml 4 g
40 ml 0.8 ml
20 ml
2 pinches in 100 ml
1M tris pH 6.8 SDS Glycerin 0.5 EDTA (pH between 7.5 und 8.5) Methanol Bromphenol blue with dH2O (short term storage 4C, long term storage at -20 C)
Stacking gel buffer 9.09 g in 50 ml
Tris-HCl dH2O dissolve adjust pH to 6.8
SDS (10%) 100 g in 900 ml
make up 1 L
SDS dH2O dissolve by heating at 37°C
SDS-running buffer (10x)
10 g 30.3 g
144.1 g in 1 L
SDS Tris Glycin H2O
TAE (Tris-Acetate-EDTA)-buffer
(50x)
242 g 57.1 ml 100 ml
Make upto 1 L
Tris Acetic Acid 0.5M EDTA with dH2O adjust pH to 8.5
TBS-buffer 4.5 g 0.71 g 0.15 g 5.5 g
2.5 ml 500 ml
NaCl Na2HPO4 NaH2PO4 Non fat dry milk Tween 20 dH2O
Transfer-buffer 1.513 g 5.63 g
Tris (25 mM) Glycin (150 mM)
Materials
30
50 ml Methanol (10 %) Adjust pH to 8.3
Resolving gel buffer 18.17 g Disslove in 100
ml
Tris-HCl dH2O adjust pH to 8.8
Trypsin-solution 10 ml 2.5 mg
Solution 1H Trypsin
Trypsin Inhibitor solution 10 ml 1.6 ml
Solution 1H Konz. D/T-I Lsg
4.6. Media and solutions for cell culture
TE671 RPMI1640 10 % 2mM 1 %
Fetal calf serum (FCS) Glutamin (200 mM) PenStrep (Penicillin/ Streptomycin)
Gibco Hyclone Gibco Gibco
C2C12 RPMI1640 10 % 2mM 1 %
Fetal calf serum (FCS) Glutamin (200 mM) PenStrep (Penicillin/ Streptomycin)
Gibco Hyclone Gibco Gibco
SKMC RPMI1640 10 % 2mM 1 %
Fetal calf serum (FCS) Glutamin (200 mM) PenStrep (Penicillin/ Streptomycin)
Gibco Hyclone Gibco Gibco
HEK 293 RPMI1640
Eahy 296 DMEM 10 % 2mM 1 %
1X 1%
Fetal calf serum (FCS) Glutamin (200 mM) PenStrep (Penicillin/ Streptomycin) HAT SodiumPyruvate
HCMEC Microvascular endothelial
Provitro
Materials
31
cell growth medium
HDMEC Endothelial Cell Growth Medium MV
Promocell
4.7. Cell lines
Name Origin Source
TE671
Rhabdomyosarcoma Prof. H. Wiendl, Würzburg
C2C12 Mouse myoblast ECCAC
SKMC Primary human
skeletal muscle cells PromoCell, Heidelberg
HEK 293 Fibroblast cells
HCMEC Human chorionic
microvascular endothelial cells
Prof. K.T. Preissner, Giessen
Eahy926 Endothelial cells Prof. K.T.Preissner, Giessen
HDMEC Human dermal microvascular
endothelial cells PromoCell, Heidelberg
4.8. Antibodies
Ab-Name against raised in Conjugation Company
GAPDH human mouse - Chemicon HLA-ABC (MHC I) human mouse - Dako IgG human rabbit AP Dako IgG human rabbit FITC Dako IgG mouse rabbit FITC Dako Isotype-control mouse FITC R&D Systems Isotyp-control mouse - Dako
Materials
32
4.9. Primers
Description
Nucleotide sequence
NCBI GenBank
PBGD 8F (fwd) 5´-TGCAACGGCGGAAGAAAAC-3´ NM_00190
PBGD 3.1 R (rev) 5´-GGCTCCGATGGTGAAGCC-3´
MHC class I(fwd) 5’ - GCTACTACAACCAGAGCGAGG - 3’ NM_002116
MHC class I(rev) 5’ - CCTCGTTCAGGGCGATGTA - 3’
LMP 2 (fwd) 5’- CGTTGTGATGGGTTCTGA - 3’ NM_148954
LMP 2 (rev) 5’ - GCAATAGCGTCTGTGGTG - 3’
LMP 7 (fwd) 5’ - TGGGGACGGAGAAAGGA - 3’ NM_148954
LMP 7 (rev) 5’ - GGCTGCCGACACTGAAAT - 3’
TAP 1 (fwd) 5’ - TGGTCTGTTGACTCCCTTACAC - 3’ NM_00593
TAP 1 (rev) 5’ - AAATACCTGTGGCTCTTGTCC - 3’
TAP 2 (fwd) 5’ - TACAACACCCGCCATCAG - 3 NM_018833
TAP 2 (rev) 5 ’- AGGTCTCTCCGCCAATACAG - 3’
4.10. Software
CellQuest® BD (FACSCalibur)
Excel 2002 Microsoft
Graph Pad Prism Software Version 4.02 (Statistical analysis + Grafics)
Microsoft Office PowerPoint 2003
Microsoft Office Word 2003
QC-Net (Unity Real Time) Bio Rad (Nephalometer analysis)
Roche Molecular Biochemicals Light Cycler Software Version 3.5 (LightCycler)
Windows 2002 Microsoft
WinMDI 2.9 (analysis of FACS data)
Methods
33
5. Methods
5.1. Cell culture
Human primary skeletal muscle cells (SKMC) (PromoCell, Heidelberg, Germany) were
cultured in skeletal muscle cell growth medium with supplements (PromoCell,
Heidelberg, Germany). Human rhabdomyosarcoma cell line (TE671) was purchased
from ATCC (Manassas, VA, USA) and cultured in RPMI Glutamax medium
supplemented with 10% FCS (fetal calf serum) and 1% penicillin-streptomycin. Mouse
C2C12 cells (ECACC) were cultured in DMEM Glutamax medium with 10% FCS and
1% penicillin-streptomycin. C2C12 cells were also differentiated to form myotubes by
culturing them in a medium containing 2% horse serum instead of FBS. The medium
used for culture of Ea.hy 926 cells (a kind gift from Department of Biochemistry,
Giessen) was DMEM containing 10 mM Hepes, 2mM L-glutamine, antibiotics (1%
penicillin-streptomycin), HAT (Hypoxanthine 100µM; aminopterin 0.4 µM; and thymidine
16 µM) 1% Na-pyruvate and 10% FCS. Human dermal microvascular endothelial cells
(HDMEC) (PromoCell, Heidelberg, Germany) were cultured in endothelial cell growth
medium MV with supplements (Promocell, Heidelberg, Germany). HCMEC cells were
grown on dishes pre-coated with collagen. The medium was endothelial cell growth
medium with supplements (PromoCell, Heidelberg, Germany). All cell lines were
maintained at 37°C and 5% CO2.
5.2. Flow cytometry
5.2.1. Autoantibody detection by flow cytometry
We recently introduced flow cytometry (FACS) to detect autoantibodies in
neuroimmunological diseases (Blaes et al. 2000). To avoid unspecific binding to
ubiquitous antigens we pre-absorbed the sera by incubating in PBS containing 1% FCS
and 0.1% NaN3 (FACS buffer) with HEK 293 cells for 24 hours at 4ºC (serum dilution
1/10) prior to incubation with the other cell cultures. The different cell lines in culture
were detached by incubation in 0.025% trypsin-EDTA (Gibco) in phosphate buffered
saline (PBS) for 1-3 minutes at room temperature, and were then washed in fresh PBS.
Methods
34
To see the binding of sera to the detached cells, cells were resuspended in FACS
buffer. Briefly, cells were washed twice in FACS buffer and then incubated with patient
or control sera (1:100) for 30 min at 4°C. Secondary antibody polyclonal rabbit anti-
human immunoglobulin FITC –conjugated (DAKO) was used in a 1:100 dilution (30 min
at 4°C in dark). Isotype matched non-binding antibodies were used as controls for
specific binding. After secondary antibody staining, cells were washed twice in FACS
buffer and analyzed in a Becton Dickinson FACScan flow cytometer (Beckton-
Dickinson, Heidelberg, Germany). Data were analyzed using CELLQUEST® software.
Mean fluorescence intensitiy (mfi) was measured for each sample. A percentage above
mean mfi + 2.5 standard deviations of the controls was considered positive.
5.2.2. MHC Class I detection
MHC class I antigen expression was determined using flow cytometry (FACS) analysis.
Briefly, cells were washed twice in PBS supplemented with 1% fetal calf serum and
0.1% sodium azide (FACS buffer) throughout the experiment and then incubated with
mouse anti-human MHC class I antibody (1:100) for 30 min at 4°C. Secondary antibody
(rabbit anti-mouse immunoglobulin FITC -conjugated) was used in a 1:100 dilution (30
min at 4°C in dark). Isotype matched non-binding antibodies were used as controls for
specific binding. After secondary antibody staining, cells were washed twice in FACS
buffer and analyzed in a Becton Dickinson FACScan flow cytometer. Data were
analyzed using CELLQUEST® software.
5.3. RNA isolation, RT-PCR, and Real time PCR
Total cellular RNA was isolated and MHC class I, LMP2, LMP7, TAP1 and TAP2
expression were analyzed by real time PCR. Primer sequences used for PCR were
designed using Primer Premier 5® software. Primer sequences are mentioned in Table
9. PBGD mRNA was used as a housekeeping gene, because its level is neither affected
by IFN-γ nor by statin treatment. A mix of the following reaction components was
prepared for each sample: 8 µl water, 1 µl forward primer (10 µM), 1 µl reverse primer
(10 µM), 10 µl QuantiFast SYBR Green PCR kit® (Qiagen, Germany) and 1 µl of
reverse transcribed mRNA sample as PCR template. Capillaries were closed,
Methods
35
centrifuged and placed into the rotor. The following general real-time PCR protocol was
used: denaturation program (10 min at 95°C), a three-segment amplification repeated
40 times (20 s at 95°C; 20 s at different annealing temperature for each primer pair; 30
s at 72°C), melting curve program (60–99°C), and finally a cooling down to 40◦C.
5.4. Cytotoxicity assay
Cytotoxicity was assessed with an lactate dehydrogenase (LDH)-cytotoxicity detection
kit (Roche Diagnostics), which measures LDH activity released from the cytosol of
damaged cells. Cells were grown in 96-well culture plates, at 37 °C, for 24 h to near
confluence. At this time they were incubated for more than 24 h in the absence or the
presence of IgGs from myositis patients or healthy controls. Cells grown in 96-well
plates were exposed to various concentrations of IgGs (50 to 800 µM) for 24 h. After 24
h, 100 µl of supernatant per well was harvested and transferred into a new 96-well, flat-
bottom plate. LDH substrate (100µl) was added to each well and incubated for 30 min at
room temperature (RT) protected from light. The absorbance of the samples was
measure at 490 nm with an ELISA reader. Cytotoxicity was calculated with the formula:
% cytotoxicity = (experimental value - low control) x 100/(high control - low control),
where low control is assay medium plus cells and high control is assay medium (plus
2% Triton X-100) plus cells.
5.5. Calcium imaging
Relative changes in the intracellular Ca2+ concentration were measured using the Ca2+ -
sensitive fluorescent dye fura-2 as described previously (Haschke, Schafer et al. 2002).
The wavelength, at which fura-2 is maximally excited, shifts in dependence of the
cytoplasmatic Ca2+ concentration. The HUVECs grown on coverslips were loaded for 60
min with 2.5 µmol/L fura-2 acetoxymethylester (fura-2/AM). Fura-2 was then washed
away. In these experiments, the cells were first superfused with tyroid solution (1
mL/min), baseline was recorded, and then control or myositis IgG were added at the
concentration of 30 mg/L, and then the effect was recorded for approximately 30 min.
Experiments were carried out at room temperature on an inverted microscope (Olympus
IX-50) equipped with an epifluorescence and an image analysis system (Till Photonics,
Methods
36
Martinsried, Germany). The emission above 470 nm was measured from several
regions of interest, each with the size of about one cell. The cells were excited
alternatively at 340 and 380 nm and the ratio of the emission signal at both excitation
wavelengths was calculated. Data were sampled at 0.33 Hz. The baseline in the
fluorescence ratio of fura-2 was measured during at least 5 min, Superfusing with a
standard or a Ca2+ free Tyrode solution (depending on the experiment), before any IgG
was administered. ATP (ATP, 50 µmol/l) was used for vitality test of cells.
Figure 6. Fura-2 loaded endothelial cells.
5.5. Protein extraction
When cells were confluent, monolayers were washed twice with 1 % PBS and then
incubated with 0.025% trypsin-EDTA (Gibco) for 1-3 minutes at room temperature to
detach them. Cells were centrifuged and then resuspended in 1 % PBS. Cells were
lysed using Dounce homogeniser and ultra sonication (3 times, 15 seconds for each).
The cell extract was centrifuged at 14,000 rpm and the supernatant was collected.
Protein quantification was performed on the supernatant using Bradford method.
Methods
37
5.5. One-dimensional gel electrophoresis (1-DE)
Proteins were separated by 1-DE on 4% to 12% precast Bis–Tris NuPAGE gels, using
MOPS running buffer (Invitrogen, Carlsbad, CA, USA). After separation, proteins were
transferred onto nitrocellulose membranes (Hybond™-c extra; GE Healthcare Life
Sciences, Piscataway, NY, USA) and stained with Ponceau red (Sigma-Aldrich).
Membranes were cut and strips were first saturated with PBS–5% dry milk, and then
incubated with patient sera (1:100 dilution) for 1 hour followed by incubation with
biotinylated conjugated mouse monoclonal anti-human IgG (Fc) (Southern
Biotechnology Associates Inc., Birmingham, AL, USA) and alkaline phosphatase-
conjugated streptavidin (CALTAG; Invitrogen), and finally revealed with NBT/BCIP
(Roche Applied Science, Indianapolis, IN, USA). Each step was followed by three
washes with PBS/Tween 0.05% buffer.
5.6. Two-dimensional gel electrophoresis (2-DE)
Adherent cells were washed with PBS and than disrupted by grinding under liquid
nitrogen in a mortar. Proteins were solubilised in sample solution (8 M urea, 2 M
thiourea, 2% pharmalyte buffer [v/v, pH 3-10]; 4% CHAPS; 30 mM DTT, 20 mM Tris).
Samples were incubated for 2 hours at 4°C, vortexed, centrifuged (18.000 × g, 30 min)
and the supernatant was subjected to isoelectric focusing (IEF) after protein
determination (BCA Protein Assay, Pierce, Rockford, IL).
2-DE was performed as described (O'Farrell 1975; Gorg, Obermaier et al. 2000).
Proteins were rehydrated over night of immobilized pH gradient (IPG) strips, 11 cm, pH
3-10 linear, (Amersham Biosciences, Freiburg, Germany). IEF of 250 - 460 µg protein
per strip was carried out in a Multiphor chamber (Amersham Biosciences) at 20°C.
Electrophoresis was carried out in SDS gels (10% or 15% acrylamide) at 25°C (600 V
for 3.5 hours) in a Hoefer SE600 (Amersham Biosciences) chamber. The gels were
either stained with Coomassie or blotted. Stained gels were scanned and molecular
weight of the proteins was calculated using the protein ladder (Invitrogen, Karlsruhe,
Germany) and pI (isoelectric point) of the proteins were determined according to the pH
value of the strips. Some gels were blotted onto polyvinylidene difluoride membrane
Methods
38
(PVDF; Hybond-P, Amersham Biosciences) by tank blotting (Hoefer Transphor,
Amersham Biosciences) using Towbin buffer with 20% ethanol for 570 Vh at 5°C. Anti-
human HRP (Dako Cytomation, Hamburg, Germany) and ECL Western Blotting System
(Amersham Biosciences) or autoradiography was used to visualize reactivities.
5.7. Protein identification by peptide mass fingerprinting
Myositis and HC sera were analyzed by western blot using 2-DE membranes. Positive
spots were excised from Coomassie stained gels and were washed once with water and
twice with 50 mM ammonium hydrogen carbonate: acetonitrile (1:1) and acetonitrile,
alternately. Gel pieces were incubated in a minimal volume of a 10 ng/µL trypsin
solution (sequencing grade, Roche Diagnostics, Mannheim, Germany) in 25 mM
ammonium hydrogen carbonate (16 h, 37°C). Peptides were extracted with 10 µL of 1%
(v/v) trifluoroacetic acid. 1 µL of the solution was mixed with 1 µL 2.5-dihidroxybenzoic
acid (20 mg/ml) in 1% phosphoric acid, 50% acetonitrile on a stainless steel target and
air dried (dried droplet). Mass fingerprints of tryptic digests were obtained by MALDI-
TOF mass spectrometry using an UltraflexTM TOF/TOF mass spectrometer (Bruker
Daltonik). Proteins were identified by database searching with peptide masses using the
MASCOT search engine. Protein identification was completed by significant Z scores or
by significant p < 0.05 probability-based Mowse Scores, respectively.
5.8. Statistical Analysis
Frequency of autoantibodies and comparison with clinical data was analysed by
Fisher’s exact test. Surface binding were analysed by two-tailed Student’s t-test
between Myositis and HC groups after validating the normal distribution of these
datasets (Kolmogorov-Smirnov test). The comparison of cytotoxicity cytotoxicity level
between different groups was done using one way ANOVA. All statistical analyses were
performed using Prism® 4.02 Software (Graph Pad Inc.). The probability level accepted
for significance was p < 0.05.
Differences between groups in flow cytometry experiments were analyzed by the
Kruskal Wallis test followed by the Mann Whitney U test for a posteriori comparison of
Methods
39
means. Results are expressed as mean of triplicates ± SD. All reported p values are
based on two-tailed statistic test, with a level of significance established at 5%.
Analyses were performed using GraphPad PRISM 4.0 (San Diego, CA).
The relative expression ratio of target gene transcripts in comparison to a reference
gene transcript was calculated based on the PCR efficiency and crossing point
deviation of each investigated transcript, as described in an in-depth report of a
mathematical model (Pfaffl 2001).
Results
40
6. Results
6.1. Presence of surface binding autoantobodies
Flow cytometry was used to show the surface binding of autoantibodies to different cell
lines (muscle endothelial and fibroblast cell lines). These cells were incubated with
1:100 diluted sera and IgG binding to the cells was measured by flow cytometry after
the application of a FITC-labelled anti-human IgG secondary antibody. Eight of 26
patients (26.9%) tested, but none of the controls showed binding to TE671 cells (t test,
p<0.0001) (Fig. 7). None of the patients or controls showed substantial binding to
SKMC, or to undifferentiated C2C12 or myotubes-differentiated (Fig. 7). Additionally,
there was no significant binding to the control cell line HEK 293 (Fig. 10). When the
resulty were analyzed based on the different myositis groups (DM, PM, OIM), all three
subgroups had elevated binding to TE671 cells compared to controls. However, there
was no difference between the myositis subgroups in surface binding to the different
muscle cell lines (Fig. 8). DM patients showed a tendency to a higher surface-binding
against C2C12 mouse muscle cells. Interestingly, the patients’ sera did not bind to the
rat cardiomyocyte cell line HL-1 (Fig. 9).
The patients have also been tested for the binding to three different endothelial cell
lines. No significant binding could be found to the HCMEC or Ea.Hy926 cells. However,
5/26 (19.2%) patients showed binding above the cut-off to HDMEC cells at a level
higher than the mean ± 2.5 standard deviations of the healthy control sera (Fig. 10).
Though DM patients were expected to show more binding compared to PM patients but
in our experiments I could not see this difference.
Results
41
Control Myositis0
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175
Bin
din
g t
o T
E671 c
ells
Control Myositis0
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200600
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o S
KM
C c
ells
Control Myositis0
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o u
ntr
eate
d C
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Bin
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o C
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e s
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d C
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Control Myositis0
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Bin
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g t
o C
2C
12 c
ells
diffe
rentiate
d w
ith
2%
hors
e s
eru
m
A
DC
B
Figure 7. Surface binding of myositis sera to different muscle cell lines (expressed as mean
fluorescence intensity) compared to healthy control sera. Cut off, marked as horizontal line, was
determined as mean of the controls + 2.5 x std. dev. Myositis patients have more binding to
TE671 cell line compared to healthy controls.
Results
42
Control OIM DM PM0
25
50
75
100
125
150
175 p<0.05
p<0.05
Bin
din
g t
o T
E671 c
ells
Controls OIM DM PM0
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500
600
700
Bin
din
g t
o u
ndiffe
rentiate
d C
2C
12
cells
Controls OIM DM PM0
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600
700
800
Bin
din
g t
o d
iffe
rentiate
d C
2C
12
cells
Controls OIM DM PM0
50
100
150
750
1000
Bin
din
g t
o S
KM
C c
ells
Figure 8. Surface binding of OIM, PM and DM sera to different muscle cell lines (expressed as
mean fluorescence intensity) compared to healthy control sera. Cut off, marked as horizontal
line, was determined as mean of the controls + 2.5 x std. dev. Myositis patients have more
binding to TE671 cell line compared to healthy controls.
Results
43
Figure 9. PM and DM patient sera have been analyzed using flow cytometry for the presence of
surface binding autoantibodies to mouse primary cardimyocytes (HL-1 cells). There is no
significant difference between the binding of control and patient sera.
Controls Myositis0
10
20
30
40
50
60B
indin
g t
o H
L-1
Card
iom
yocyte
s
Results
44
Control Myositis0
50
100
250
350
Bin
din
g t
o H
CM
EC
endoth
elial
cells
Control Myositis0
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g t
o H
DM
EC
endoth
elial
cells
Control Myositis0
25
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75
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g t
o E
aH
y296 e
ndoth
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l
cells
Control Myositis0
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Bin
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g t
o H
EK
293 c
ells
A
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o H
CM
EC
endoth
elial
cells
Control Myositis0
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endoth
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Control Myositis0
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45
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55
Bin
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o H
EK
293 c
ells
A
DC
B
Figure 10. Surface binding of myositis sera to different endothelial cell lines (expressed as mean
fluorescence intensity) compared to healthy control sera. Cut off, marked as horizontal line, was
determined as mean of the controls + 2.5 x std. dev. Myositis patients have more binding to
HDMEC cell line compared to healthy controls.
Results
45
Uns
timulat
ed
Lova
stat
in
Mev
asta
tin
Simva
stat
in
0
100
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400
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1000
Bin
din
g t
o s
tatin t
reate
d S
KM
C c
ells
P<0.001
P<0.001
Uns
timulat
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Lova
stat
in
Mev
asta
tin
Simva
stat
in
0
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600
1000
Bin
din
g t
o s
tatin t
reate
d S
KM
C c
ells
P<0.001
P<0.001
Uns
timulat
ed
lova
stat
in
Mev
asta
tin
Simva
stat
in
0
100
200
300
400
500500
750
1000p<0.05
p<0.001
p<0.001
Bin
din
g t
o s
tatin t
reate
d T
E671 c
ells
In a recent report statins have been implicated to trigger myositis. Therefore, muscle
cell lines (TE671 and SKMC) were treated with different statins (Lovastatin, Mevastatin
and Simvastatin) in combination with IFN-γ to increase the immunogenecity of the
muscle cells. These cells were then used to analyze the binding of myositis sera using
flow cytometry. In TE671 cells, only the treatment with mevastatin led to an increased
binding of myositis sera, whereas the binding to simvastatin was even lower than to
unstimulated TE671 (p<0.001 and p<0.05). Using the primary muscle cell line SKMC,
Mevastatin- and Simvastatin-treated cells show significant higher binding of myositis
sera (Fig. 11). The different patient groups (OIM, DM, and PM) were then analyzed. DM
patients showed higher binding to simvastatin-stimulated SKMC compared to OIM and
PM patients (p<0.05 and p<0.01, Fig. 12) There was no difference between different
patients in binding to Mevastatin treated SKMC cells (Fig. 12).
Figure 11. Surface binding of PM and DM patient sera to statin (Lovastatin, Mevastatin and
Simvastatin) treated TE671 and SKMC cells. Cells were first treated with different statins for 48
Hrs and then analyzed by flow cytometry for binding of autoantibodies to these cells.
Results
46
Figure 12. Surface binding of PM and DM patient sera to statin (Lovastatin, Mevastatin and
Simvastatin) treated TE671 and SKMC cells. Cells were first treated with different statins for 48
Hrs and then analyzed by flow cytometry for binding of autoantibodies to these cells.
OIM DM PM OIM DM PM OIM DM PM0
100
200
300
400
500500
750
1000
Untreated Simvastatin Mevastatin
p<0.05
p<0.01
Bin
din
g t
o S
KM
C c
ells
Results
47
6.2. Western blotting of autoantibodies with muscle cell extract
Next the myositis sera were checked for their binding to muscle cell protein using
western blotting. As is shown in Fig. 13, patient sera recognized a protein in the size
range of 97-120 KDa which was absent in the fibroblast cell lysate. It again shows that
PM and DM patients do have muscle antigen specific autoantibodies.
Figure 13. Immunoblot incubated with the patient’s serum on TE671 cells (A) and on HEK 293
(B) as non-muscle control. Cell lysate was prepared from muscle and fibroblast cells and run
onto a 4-12% gradient SDS-PAGE. After blotting of proteins to nitrocellulose membrane, the
membranes were incubated with patients and healthy control sera and then the binding was
visualized by using enzyme conjugated secondary antibody and NBT substrate. Line 1 to 15
represents health controls and line 16 to 40 patients. Patient sera show a specific reactivity, to
proteins from muscle cell lysate, in range of 97-112 KDa ((A) inside the rectangle) which is
absent in blot of fibroblast cell lysate (B).
Results
48
6.3. Identification of autoantigens in muscle cells
Our experiments have shown the presence of surface binding autoantibodies in myositis
patients. To see if they bind to intracellular antigens, western blotting was performed
using the cell extract from muscle cells. It has been found that 10 out of 25 (40.0%)
myositis patients show a specific band, against muscle cell lysate, between 95-110 KDa
(Fig. 13 see the boxed area), compared to none aginst fibroblast cell lysate ( 5 DM, 1
PM, and 4 OIM).
In order to identify muscle proteins targeted by serum IgG from myositis patients,
muscle proteins from TE671 cells were submitted to 2-DE and transferred to PVDF
membranes (Fig. 14). Serum IgGs from 4 different myositis patients and a healthy
control individual were incubated with the PVDF membranes transferred with muscle
proteins.
Among many spots transferred to the PVDF membranes, 60 or so spots were
recognized by serum IgG from patients and controls. Only the spots which showed
immunoreactivity with patients’ serum IgGs were submitted to MALDI-TOF mass
spectrometric analysis. These spots were identified as glycyl-tRNA synthetase, aldose
reductase, Proteasome subunit beta type 7 precursor and others (Table 3).
Results
49
Figure 14. 2D-PAGE blots of cell lysate from TE671 cells with healthy control (A) and patient
(B). Cell lysate was prepared and first applied to isoelectric focussing and then the isoelectric
strips were placed onto SDS-PAGE for second dimensional electrophoresis. After the
electrophoresis the protein were blotted to PVDF membranes and these membranes were then
probed with either control (A) or myositis (B) sera.
Results
50
Spot-Nr.
Protein Accession number Human
M (kDa) exp.
M (kDa) theor
.
pI exp.
pI theor
.
Mowse score (>55)
Seq. cov. (%)
Number of ident. peptides
1 glycyl-tRNA synthetase
SYG 75 83.8 5.7 6.61 206 38 28
2 Annexin A2 + Aldose reductase
ANXA2
ALDR
35 38.8
7.0 7.57
350 162 160
54
72
20
22
3 Proteasome subunit beta type 7 precursor + Platelet-activating factor acetylhydrolase IB subunit beta
PSB7
PA1B2
28 30.3
25.7
5.5 7.57
5.57
259 143
109
59
47
18
12
4 Proteasome subunit beta type 7 precursor
PSB7 28 30.3 5.6 7.57 155 57 16
Blank ___
-- --
BSA BSA
-- -- 144
Table 3. Summary of proteins identified from the spots picked up from 2D gel after
immunoblotting.
6.4. Cytotoxic effect of autoantibodies
To measure the cytotoxic effect of different patients’ IgG on various cell lines, cells were
incubated with purified IgGs in 96 well plates for 24 hours and later the cytotoxicity was
measured using LDH cytotoxicity assay kit. Before incubating the cells with IgGs, first
cells were plated at different densities for 24 hours in 1% FBS medium and then
incubated with IgGs to find out the right cell density for further experiments. It has been
deduced that 10,000 cells per well is the optimum cell density which has a linear
function in the cytotoxicity assay. Ten thousands cells per well were then incubated with
various concentrations of purified IgGs (50 to 800 µM), and it was found that for some
cells the effective IgG concentration was 10 ug/ml and for some 50 ug/ml. The above
density of cells and appropriate concentration of IgGs was then used for further
Results
51
experiments. The results obtained after 24 hours incubation of IgGs with different cell
lines are summarised in (Fig. 15). The cytotoxic effects of myositis IgG was visible only
against the endothelial cell lines Ea.Hy and HDMEC (student’s t test) p<0.05. Though
surface binding antibodies have been found in myositis sera against TE671, which is an
established model cell line for muscles, I could not see any cytotoxicity in this cell line.
None of the healthy controls showed any cytotoxicity (Fig. 15).
Results
52
Figure 15. Purified IgGs from PM and DM patients have been tested with endothelial and
muscle cell lines for their cytotoxic effects using LDH assay. Cells were incubated with IgGs for
24 hours and then supernatant was used to detect cytotoxicity using LDH cytotoxicity assay.
Control Myositis
-50
0
50
100
150
200
% c
yto
toxic
ity a
gain
st
TE
671
control Myositis0
2
4
6
8
10
12
% c
yto
toxic
ity a
gain
st
SK
MC
Control Myositis
-0.050
-0.025
0.000
0.025
0.050
% c
yto
toxic
ity a
gain
st
HE
K2
93
control Myositis-0.005
-0.004
-0.003
-0.002
-0.001
0.000
p<0.0001
% c
yto
toxic
ity a
gain
st
EaH
y296
Control Myositis0
10
20
30
40
50
60
70
80p=0.0109
% c
yto
toxic
ity a
gain
st
HD
ME
C
Results
53
6.5. Effect of autoantibodies on endothelial calcium flux
As has already been shown that IgG from some of the patients show cytotoxicity against
endothelial cells, it becomes necessary to check if this cytotoxic effect is due to change
in Ca2+ influx. To investigate this effect HUVEC cells, which are an established model
for Ca2+ measurement, were used. The cells were cultured on coverglass and then
mounted on the microscope in Ca2+ free medium. The IgG were then added to the cells
after monitoring the cells for 5 minutes without adding anything. Two myositis patients
IgG (1 DM and 1 PM) and a control IgG were used for the experiment. It has been found
that HUVEC cells show a significant change in calcium influx after addition of IgGs from
myositis patients while healthy control IgG failed to induce any change in calcium influx
(Fig. 16).
50
100
150
200
0 5 10 15 20 25 30
Time
Calc
ium
Ratio
Blank
HC
P1
P2
50
100
150
200
0 5 10 15 20 25 30
Time
Calc
ium
Ratio
Blank
HC
P1
P2
Blank
HC
P1
P2
Figure 16. Purified IgGs (3g/L) were added to the primary HUVEC cells and then Ca2+
influx
was measured using image analysis system (Till Photonics, Martinsried, Germany).
Results
54
6.6. Effects of statins on muscles cells
In surface binding experiments using flow cytometry, it could be shown that statins
treatment of SKMC cells do indeed affect the surface binding of myositis sera to these
cells (p<0.001) (Fig. 11). The very obvious difference was seen in mevastatin (p<0.01)
and simvastain (p<0.01) treated cells, but lovastatin treatment did not show any
increase in surface binding. However, in case of TE671 cells no difference could be
seen, infact simvastatin treated TE671 show less surface binding to myositis sera even
compared to controls (Fig. 11)
Statins, have been prescribed extensively for their cholesterol-lowering properties and
efficacy in cardiovascular diseases (Greenwood et al. 2006). Statins also have
additional immunomodulatory properties that operate independently of lipid lowering. In
some patients, statins can induce necrotizing or inflammatory myopathies (Needham et
al. 2007). Increased MHC class I expression has been shown in muscle biopsies of
statin-induced myopathy. Therefore, the effect of statins on the expression of MHC
class I in muscle cells was investigated in order to evaluate the hypothesis of an altered
immunogenecity of muscle cells induced by statins.
6.6.1. Statins enhance IFN-γ-induced MHC class I expression in TE671 but not in SKMC
The effect of statins on the MHC class I expression was analyzed in two different
muscle cell lines – the established cell line TE671 and the primary cell line SKMC.
Additionally, all three statins (Lovastatin, Mevastatin and Simvastatin) were also tested
for their ability to affect IFN-γ-induced MHC class I expression in TE671 and SKMC cell
lines. First, cells were treated with different concentrations of statins to see if they show
any dose dependent effect on MHC class I expression, but they failed to show any dose
dependency (Fig. 18). Therefore 10 µM concentrations of statins was used in further
experiments, as it has been cited in other reports as well. Cells were treated with statins
(10µM) and/ or with IFN-γ (100ng/ml) for 48 hours, and MHC class I expression was
examined using flow cytometry.
SKMC and TE671 in the untreated condition expressed MHC class I (Fig. 19 and 20).
Results
55
10 0 10 1 10 2 10 3 10 4
Empty
032
I
Eve
nts
MHC Class I expression
Statin
DMSO
IFN-γ
IFN-γ+Statin
32
Eve
nts
-
MHC Class I expression
IFN-γ
IFN-γ+Statin
Statin
DMSO
10 0 10 1 10 2 10 3 10 4
Empty
032
I
Eve
nts
MHC Class I expression
Statin
DMSO
IFN-γ
IFN-γ+Statin
10 0 10 1 10 2 10 3 10 4
Empty
032
I
10 0 10 1 10 2 10 3 10 4
Empty
032
10 0 10 1 10 2 10 3 10 4
Empty
032
I
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nts
MHC Class I expression
Statin
DMSO
IFN-γ
IFN-γ+Statin
32
Eve
nts
-
MHC Class I expression
IFN-γ
IFN-γ+Statin
Statin
DMSO
32
Eve
nts
-
MHC Class I expression
IFN-γ
IFN-γ+Statin
Statin
DMSO
32
Eve
nts
-
MHC Class I expression
IFN-γ
IFN-γ+Statin
Statin
DMSO
32
Eve
nts
-
MHC Class I expression
32
Eve
nts
-
MHC Class I expression
IFN-γ
IFN-γ+Statin
Statin
DMSO
IFN-γ showed an induction of MHC class I expression in both cell lines (MFI 781 vs. 312
in controls in SKMC; MFI 85 vs. 28 in TE671). Surprisingly, statins alone induced a
significant reduction of the baseline expression of MHC class I in primary muscle cells
(SKMC); however, they had no effect in TE671 (Fig. 19 and 20).
Statins alter the IFN-γ-induced expression of MHC class I differently in SKMC and in
TE671. Statins significantly reduced the IFN-γ-induced MHC class I expression in
primary muscle cells (SKMC) (a decrease in MFI from 781 to 312) while they promoted
a robust opposite effect in TE671 (an increase in MFI from 85 to 200) (Fig. 19 and 20).
Further, this effect has been checked at the RNA level, and it was found to complement
the protein level. I could detect an increase in MHC class I gene expression after IFN-γ
treatment in SKMC (17.8 fold) and TE671 (8.9 fold) (Fig. 21). The expression of MHC
class I was not substantially affected by any statin treatment in any of the cell lines. This
highlights the ineffectiveness of statins to enhance the MHC class I expression in vitro.
A. B.
Figure 17. Effect of statins on MHC class I expression accessed by flow cytometry. (A) and (B)
are representative flow cytometry analysis of MHC class I expression following different
treatments of SKMC and TE671, respectively.
Results
56
Figure 18. Effect of different concentrations of statins on MHC class I surface expression.
SKMC and TE671 cells were treated with different concentrations of statins ranging from 10 nM
to 10 µM.
On the other hand, a 53-fold increase in MHC class I gene expression on average was
detected when SKMC cells were treated with statins along with IFN-γ (Fig. 21). This
effect could nevertheless not be seen at the protein level, which might indicate possible
involvement of a translational regulation of MHC class I in primary cells. A positive effect
was also exhibited in the case of simvastatin and slightly in the case of lovastatin on
IFN-γ-induced expression of MHC class I in the TE671 cell line, in accordance with the
FACS data (Fig. 21).
Results
57
Figure 19. Effect of statins on MHC class I expression assessed by flow cytometry. (A, B, C)
SKMC cells treated with IFN-γ (100ng/ml), statins (10µM) or statins along with IFN-γ.
Treatment with statins reduced significantly MHC class I expression and IFN-γ inducible MHC
class I expression in SKMC. (A) Treatment with Lovastatin; (B) treatment with Mevastatin; and
(C) treatment with Simvastatin. Data are represented as mean of triplicates ± SD. *Values
significantly different from corresponding treatment (Kruskal Wallis test followed by the Mann
Whitney test, p < 0.05).
Results
58
Figure 20. Effect of statins on MHC class I expression accessed by flow cytometry. (A, B, C)
TE671 cell line treated with IFN-γ (100ng/ml), statins (10µM) or statins along with IFN-γ.
Statins showed differential effects in two different cell lines. Treatment with statins reduced
significantly MHC class I expression and IFN-γ inducible MHC class I expression in SKMC.
Same treatment here showed no considerable effect on MHC class I expression although
conversely enhanced IFN-γ-induced MHC class I expression in TE671. (A) Treatment with
Lovastatin; (B) treatment with Mevastatin; and (C) treatment with Simvastatin. Data are
represented as mean of triplicates ± SD. *Values significantly different from corresponding
treatment (Kruskal Wallis test followed by the Mann Whitney test, p < 0.05).
Results
59
Figure 21. Effect of statins on MHC class I expression at transcriptional level accessed by real-
time PCR in (A) SKMC cells and (B) TE671 cells. The graphic shows the fold induction level
of MHC class I expression compared to untreated cells. These values are representative of three
experiments done in triplicate.
Results
60
6.6.2. TAP and LMP expression
The assembly of MHC class I molecules with peptides is orchestrated by several
factors, including the transporter associated with antigen processing (TAP) (Raghavan
et al. 2008). TAP1 and TAP2 gene products are responsible for the transport of the
peptides from the cytosol to the endoplasmic reticulum, where peptides bind to newly
sythesized MHC class I (Elliott 1997). The low- molecular-mass polypeptides 2 (LMP2)
and 7 (LMP 7) increase the amount of generated peptides available for binding to MHC
class I molecules (Driscoll et al. 1993; Gaczynska et al. 1994; Groettrup et al. 1995;
Niedermann et al. 1995). Up-regulation of the TAP and LMP genes by immune
regulators, such as interferon-gamma (IFN-γ), leads to an increase in MHC class I
expression (Yang et al. 1992; Boes et al. 1994; Tanaka 1994). Determination of TAP1,
TAP2, LMP2 and LMP7 expression was done at RNA level after 48 h of treatment with
IFN-γ, statins, or a combination of both. IFN-γ treatment increased the expression of all
four genes (TAP1, TAP2, LMP2 and LMP7) (Table4). Statins alone increased the
expression level of TAP1 (5 fold) and LMP7 (2.8 fold) in SKMC cells, but they failed to
show any effect in TE671. No significant change could be detected in other genes
evaluated in response to statin treatment. All three statins potentiated the IFN-γ
induction of TAP2, LMP2 and LMP7 in SKMC but not in TE671 (Table 4).
Results
61
GENE / TREATMENT IFN-γ lova meva simva IFN-γ
+ lova
IFN-γ +
meva
IFN-γ +
simva
SKMC LMP2 17.83 1.29 0.94 1,10 30.21 36.03 37.09
LMP7 9.45 2.40 1.77 2.87 11.62 16.22 16.35
TAP1 28.03 4.81 2.53 5.06 90.82 89.21 123.37
TAP2 8.47 0.33 0.71 0.27 16.53 10.47 18.83
TE671 LMP2 123.05 0.66 0.61 0.60 97.40 101.19 104.91
LMP7 23.87 0.78 0.85 0.59 23.84 25.40 8.51
TAP1 53.77 0.80 0.73 1.34 53.86 49.13 38.61
TAP2 7.96 0.72 0.41 0.82 6.24 9.31 5.09
Table 4. Relative gene expression analysis by quantitative real time PCR of genes involved in
MHC class I antigen presentation after statins and/or IFN-γ treatment in muscle cell lines.
Discussion
62
7. Discussion
In this thesis, a combination of different autoantibody detection methods and protein
biochemistry methods, such as 2D-gelelectrophoresis and mass spectrometry has been
used to detect muscle- or muscle-endothelium-specific autoantigens and their
functionality in inflammatory myopathies. The identification of such antigens could
contribute significantly to elucidate the physiopathology of these diseases.
7.1. Surface binding autoantibodies
It could be shown that a significant number of myositis patients have autoantibodies
against surface epitopes of muscle cells. This binding was specific to muscle cells since
there was no reactivity either in control sera or against fibroblasts. At the same time
there was no surface binding to C2C12 mouse muscle cell line either undifferentiated or
differentiated or to primary muscle cells SKMC. This might be due to a species
specificity of the autoantibodies (Katsumata et al. 2007) and the differences between
the primary cells and rhabdomyosarcoma cells. Previous studies have reported that
over 50% of the patients have autoantibodies diected against intracellular antigens,
although with improved detection techniques, recent studies have shown this frequency
to be nearer 80% (Gunawardena et al. 2009 a). These autoantibodies can be
categorized into myositis-associated autoantibodies (MAAs) and myositis-specific
autoantibodies (MSAs). The MAAs, anti-U1-RNP, anti-U3-RNP, anti-PM-Scl and anti-
Ku, are principally seen in myositis-scleroderma overlap syndromes (Koenig et al.
2007), whereas the MSAs (anti-ARS, anti-SRP, anti-Mi-2, and anti-SAE etc.) are highly
selective, mutually exclusive and are associated with particular genotypes and clinical
phenotypes within the myositis spectrum (Love et al. 1991; Chinoy et al. 2006; O'Hanlon
et al. 2006; Gunawardena et al. 2009 a). MSAs target either nuclear or cytoplasmic
components of the cell that are involved in gene transcription, protein translocation and
antiviral responses. In a recent study Gunawardena et al. have discussed the use of
MSAs to define patients into clinical syndromes, which might help in predicting the
outcomes and influence the treatment strategies (Gunawardena et al. 2008). Though in
our experiments these surface binding autoantibodies could not differentiate among
Discussion
63
different myositis groups (DM, PM and OIM) we did see that DM patients showed an
increased affinity towards undifferentiated C2C12 mouse muscle cells. The question of
whether autoantibodies are simply epiphenomena or directly linked to pathogenesis
remains unclear. Several attractive paradigms have been proposed to explain why
certain intracellular proteins are selectively targeted by autoantibodies (Helmers et al.
2009; Gunawardena et al. 2009 a).
Statin treatment of muscle cells increased the binding of myositis sera to these cells
depending on the different statins used and the cell lines tested. This effect was more
pronounced in primary muscle cells SKMC, where mevastatin and simvastatin treated
cells showed significantly increased binding to myositis sera compared to untreated
cells. DM patients showed the highest binding to simvastatin treated primary muscle
cell. This clearly indicates that statins definitely play a role in statins induced myositis.
The availability of MSAs for classifying the patients does not help much in predicting the
outcome of disease (Gunawardena et al. 2009 a). The future development of assays
that test for MSAs in routine clinical practice is important. Investigating the structure and
function of target molecules, and whether autoantibodies themselves have functional
roles appears critical for understanding the pathogenic mechanisms in this complex
spectrum of diseases. Though these disorders are believed to be mediated by cellular
immune mechanisms, several investigators have identified immunoglobulin and
complement in the muscles (Whitaker and Engel 1972; Kissel et al. 1986) and focused
on vascular deposition of autoantibodies in relation to pathogenesis. Pathogenic role of
anti endothelial cell antibodies (AECA) have been implicated in several diseases
including autoimmune rheumatic diseases and (Domiciano et al. 2009; Jarius et al.
2009). Konstantin et al. developed a cellular ELISA to detect AECA in patients with
IIMs. Fourteen of 19 patients (77%) were shown to have EC-binding activities. Yet, the
main message from their study was that EC-specific IgG autoantibodies were more
often encountered in MCTD and SS-associated myositis than in idiopathic PM and DM
(Salojin et al. 1997). This group contemplated that AECA might be pathogenic through
complement activation, antibody dependent cell cytotoxicity, or apoptosis of EC (Cines
et al. 1984; Meroni 1994; Bordron et al. 1998). As the involvement of the endothelium is
Discussion
64
suggested in the pathogenesis of these IIMs, especially in DM (Nagaraju et al. 2006), I
also looked for the presence of anti endothelial cell antibodies. Any surface binding
antibodies could not be shown to the endothelial cell lines tested, yet surprisingly these
antibodies showed cytotoxicity against endothelial cells. In Wegener’s granulomatosis
(WG), Holmen et al. showed that WG IgG bind strongly to HKMEC but not to HUVEC
and other endothelial cell lines (Holmen et al. 2007), which shows that these
autoantibodies are specific for kidney EC and not just any EC. It seems that in our
experiments also these autoantibodies are very specific, but then again any surface
binding to human dermal micro-vascular endothelial cells (HDMEC) could not be shown.
7.2. Proteomic approach to identify autoantigens
In IIMs, like most autoimmune diseases, several autoantigens participate in the
pathogenesis, and epitope spreading is accountable for disease induction, progression
and inflammatory relapses. Knowledge of the targeted autoantigens is therefore
indispensable for understanding the nature of acute autoimmune responses during
recurrent attacks. Furthermore identifying persons at risk to develop several
autoimmune diseases is based upon the presence of an autoreactive response to
several autoantigens prior to disease state (Verge et al. 1996; LaGasse et al. 2002;
Maclaren et al. 2003; Scofield 2004). The accuracy of prediction increases by
combining autoantigens to identify potential novel candidate autoantigens in IIMs. In
current study the IgG binding profile to the muscle cells proteome was tested.
A proteomic approach based on 2D immunoblotting was used, which combines 2D
electrophoresis and immunoblotting. The 2D electrophoresis is a classical method for
proteome analysis. It is a powerful tool to investigate differential patterns of qualitative
and quantitative protein expression. Theoretically, all the proteins in the cells are
separated in one gel. In the present method, 2D immunoblotting was performed and
analysis of the western blot images was done with computer systems. The matching
procedure allows determining the precise localization of relevant protein spots.
Thereafter the selected protein spots were identified by mass spectrometry. Thus, this
technical approach of 2D immunoblotting with mass spectrometry also called
Discussion
65
“serological proteome analysis” (SERPA) potentially enables the detection and
identification of new target antigens. So far, proteome-based technologies have been
successfully used in tumor immunology for the identification of tumor-specific
autoantigens (Seliger and Kellner 2002; Unwin et al. 2003). In the very last years, 2D
immunoblotting with mass spectrometry have been used to identify new antigens
targeted by autoantibodies, including myelin protein Po in autoimmune inner ear
disease (Cao et al. 1996), heterogeneous nuclear ribbonucleoprotein A2/B1 in
autoimmune hepatitis,(Huguet et al. 2004) ATP synthase beta chain in celiac
disease,(Stulik et al. 2003) and α-enolase in Behcet’s disease (Lee et al. 2003). In
rheumatoid arthritis, autoantibodies directed against α-enolase and its citrullinated
molecule have also been detected (Saulot et al. 2002; Kinloch et al. 2005). Another
approach, the SEREX method (serological analysis of recombinant cDNA expression
library), has been developed for the serological definition of immunogenic tumor
antigens (Chen et al. 1997; Fernandez Madrid et al. 2005; Kuboshima et al. 2006).
Recent studies indicate that the SEREX approach may also be utilized for the analysis
of complex immune responses involved in autoimmune diseases (Krebs et al. 2003).
Whereas human proteins are directly used as source of antigens in 2D
immunoblotting, proteins are expressed in E. coli in SEREX approach. Although several
improvements can be made in the SEREX approach (Fernandez Madrid et al. 2005),
the use of human cells as a direct source of proteins seem to us more straightforward
and relevant for researches in autoimmune diseases than using proteins expressed in
E.Coli.
Sera from 26 patients and 15 healthy subjects were analyzed by western
immunoblotting with total cell lysate from TE671, C2C12 and HEK 293 cell lines.
Seropositivity was based on immunoreactivity with different proteins at serum dilutions
of 1/100. With TE671 cell lysate, 12 sera from 26 (46%) myositis patients produced a
specific band in the region between 97 and 191 KDa which was not present in healthy
controls. This immunoreactivity failed to appear with HEK 293 (Fig. 13) and therefore,
was considered to be muscle specific.
These results further show the presence of a specific protein band in western blotting of
Discussion
66
muscle cell lysate with patient sera which was not present in control sera and fibroblast
cell lysate. Though specific spots in 2-D electrophoresis of muscle cell proteins were
found, yet these proteins are not muscle specific instead ubiquitously expressed. The
identified proteins include Proteasome subunit beta type 7 precursor proteins. Anti-
proteasome antibodies have already been reported in various diseases including SLE,
primary sjögren’s syndrome, PM and DM (Scheffler et al. 2008). They have also
discussed that the autoimmune response against proteasomes is diverse and not
disease specific, and due to frequent and extended antibody reactivity, the proteasome
is one of the prominent autoantigens in systemic autoimmune disorders. The
autoantigens themselves might play a role in disease mechanisms. Enhanced
expression of Mi-2 and Jo-1 in myositis muscle compared with normal muscle, that too
primarily in regenerating muscles rather than in mature myotubes very nicely supports
the hypothesis that the presence of myositis candidate autoantigens during reparative
myogenesis can drive induction and propagation of the autoimmune response
(Casciola-Rosen et al. 2005). Mi-2, which is essential for the development and repair of
the basal epidermis (Kashiwagi et al. 2007), is preferentially expressed in DM muscle
rather than PM muscle, supporting the association between DM and Mi-2 (Casciola-
Rosen et al. 2005). Certain autoantigenic-tRNA synthetases (histidyl, asparaginyl and
tyrosyl) have chemoattractant properties and can induce leukocyte migration through
the CCR5 and CCR3 receptors (Howard et al. 2002; Wakasugi et al. 2002).
As the involvement of the endothelium is suggested in the pathogenesis of these
diseases (Nagaraju et al. 2006), the presence of anti endothelial cell antibodies has also
been analyzed. Any surface binding antibodies could not be shown to any of the
endothelial cell lines tested, yet surprisingly these antibodies showed cytotoxicity
against endothelial cells.
7.3. Functional effects of autoantibodies
Functional autoantibodies that act at ion channels or receptors and disrupt autonomic or
cardiovascular functions have been described in autoimmune diseases including
Lambert-Eaton Myasthenic syndrome (LEMS) (Waterman et al. 1997), Scleroderma
Discussion
67
(Goldblatt et al. 2002), Sjögren’s syndrome (Waterman et al. 2000), and others
(Gleicher et al. 2007; Jackson et al. 2008; Maciejewska-Rodrigues et al. 2010). LEMS
IgG impairs transmitter release from parasypathetic and sympathetic neurons through
down-regulation of one or more subtypes of voltage-gated calcium channels (Waterman
et al. 1997). The IgG from patients with primary Sjögren’s syndrome show two effects
on the submandibular glands. First, it may act as an inducer of the proinflammatory
molecule (PGE2), which then inhibits Na+/K+ -ATPase activity. Secondly, it may be
involved in dry mouth pathogenesis by abolishing the Na+/K+ -ATPase inhibition and the
net K+ efflux stimulation of the salivary glands (Passafaro et al. 2010).
Since disturbances of cell functions often lead to an increased cell death rate, possible
cytotoxicity of myositis-IgG on muscle and endothelial cells has been investigated.
These autoantibodies showed no surface binding to any of the endothelial cells, but
they did show their cytotoxic effects against these cells. Since binding of obviously
functional autoantibodies it could not be shown, one might speculate that the antigens
responsible for the cytotoxic effect are expressed at low level on the cell surface.
Another explanation could be that, the autoantibodies are low-affinity binding IgGs and
our method (flow cytometry) is not able to discriminate the specific binding from
background. Low-affinity binding autoantibodies have also been recently found in
patients with myasthenia gravis where no anti-AChR antibodies could be found in
normal tests (Leite et al. 2008).
Calcium is an important messenger in intracellular signalling mechanisms; therefore it
has been contemplated that the cytotoxic antibodies might have an effect on stimulated
calcium flux. The results show that the purified IgG from patients indeed triggered a
rapid calcium flux response in HUVECs while healthy control IgG did not show any
effect at all. This change in Ca2+ might be responsible for the cytotoxic effects of
antibodies.
HUVECs are a well established model to study the changes in calcium flux. A change in
calcium flux is the first feature of cell activation, which initiates and coordinates specific
cellular activities after a given stimuli. Calcium is an important intracellular messenger
Discussion
68
that is involved in modulating a vast array of cellular events. Signalling events through
the BCR in SLE B cells are abnormal, as indicated by increased intracellular calcium
flux and phosphorylation of multiple proteins. Stimulation of freshly isolated peripheral
blood B cells from patients with SLE with BCR ligand led to unusually high calcium
responses and increased tyrosine phosphorylation of proteins relative to peripheral
blood B cells from healthy individuals and disease controls (Pugh-Bernard et al. 2006).
The signalling alterations found in this study did not correlate with disease activity or
treatment, and clearly demonstrate the existence of SLE-specific signalling alterations in
B cells.
7.4. Skeletal muscle cell MHC I expression in response to statin treatment
The major histocompatibility complex (MHC) genes are located on chromosome 17 (in
the case of H-2) and 6 (human leukocyte antigen [HLA]) in mice and humans,
respectively. MHC class I molecules are composed of a transmembrane heavy-chain
glycoprotein (H), a non-covalently associated soluble protein called ß2-microglobulin,
and a short peptide of 8-10 residues derived from endogenous proteins. These genes
are constitutively expressed in most adult tissues, although the relative levels of class I
expression in different tissues vary widely.
MHC class I over expression is an early event in many autoimmune diseases,
particularly in tissues such as muscle, pancreatic ß cells, neuronal cells, and thyrocytes
that show little or no constitutive expression (Hanafusa et al. 1983; Bottazzo et al. 1985;
Foulis et al. 1987; McDouall et al. 1989). Abnormal high expression of these molecules
can occur in the absence of an inflammatory infiltrate, suggesting that it may be
independent of, and possibly precede, the effects of cytokines released from infiltrating
mononuclear cells. Normal human skeletal myoblasts constitutively express low levels
of HLA class I molecules under cell culture conditions (Hohlfeld and Engel 1991;
Nagaraju et al. 1998). Muscle fibres in mormal individuals do not express detectable
levels of MHC class I antigens (Emslie-Smith et al. 1989; Hohlfeld and Engel 1991).
Muscle fibres from patients with IIMs show consistently strong expression of MHC class
I molecules (Rowe et al. 1983; Appleyard et al. 1985; Karpati et al. 1988; Emslie-Smith
Discussion
69
et al. 1989; McDouall et al. 1989; Bartoccioni et al. 1994). Class I MHC may play
multiple roles in myositis serving both to initiate the disease as well as maintain ongoing
muscle damage. A big part of IIM immunogenetic research has continued to
concentrate on the MHC. In IIM it has long been recognized that HLA status relates to
disease specific serologivcal subtypes, which in turn associate with distinct clinical
phenotypes (Love et al. 1991).
The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, generically referred to
as statins, have been prescribed extensively for their cholesterol lowering properties
and efficacy in cardiovascular diseases (Greenwood et al. 2006). Statins also have
additional immunomodulatory properties that operate independently of lipid lowering. In
some patients, statins can induce necrotizing or inflammatory myopathies (Needham et
al. 2007). However, basic mechanisms governing this property are so far unknown.
Recently, increased MHC class I expression has been demonstrated in muscle fibers of
patients afflicted with statin-induced myopathies. The over-expression of MHC class I
molecules is an early event in many autoimmune diseases, since it is a prerequisite for
the cytolytic action of cytotoxic T lymphocytes. MHC class I molecules by themselves
can have a deleterious effect on cell types that do not constitutively express these
molecules (Nagaraju 2005). Normal human skeletal myoblasts constitutively express
low levels of MHC class I molecules under cell culture conditions (Hohlfeld and Engel
1991; Nagaraju et al. 1998). Muscle fibres of healthy individuals do not express
detectable levels of MHC class I antigens, although these fibres have been shown to
express MHC class I in several autoimmune muscle diseases (Appleyard et al. 1985;
Emslie-Smith et al. 1989).
The assembly of MHC class I molecules with peptides is orchestrated by several factors
including the transporter associated with antigen processing (TAP)(Raghavan et al.
2008). TAP1 and TAP2 gene products are responsible for the transport of peptides from
the cytosol to the endoplasmic reticulum where peptides bind to newly synthesized
MHC class I (Elliott 1997). The low molecular mass polypeptides 2 (LMP2) and 7
(LMP7) increase the amount of generated peptides available for binding to MHC class I
antigens (Driscoll et al. 1993; Gaczynska et al. 1994; Groettrup et al. 1995; Niedermann
Discussion
70
et al. 1995). Up-regulation of the TAP and LMP genes by immune regulators, such as
interferon gamma (IFN-γ) lead to an increase in MHC class I expression (Yang et al.
1992; Boes et al. 1994; Tanaka 1994).
The mechanism responsible for widespread expression of MHC class I in statin-
associated myopathy is unclear (Needham et al. 2007). Therefore, the effect of statins
on the MHC class I expression has been investigated in vitro on primary cultured
muscle cells and the rhabdomyosarcoma cell line TE671, which is often used as a
model cell line for skeletal muscle diseases. Surprisingly, statins impaired the IFN-γ-
inducible MHC class I expression in primary skeletal muscle cells SKMC while they
potentiate the same effect in TE671. In primary skeletal muscle cells, statins alone
showed even a decrease in surface expression of MHC class I.
The potential effect of three statins has also been investigated on MHC class I
expression and regulation in response to IFN-γ in primary skeletal muscle cell cultures
and the rhabdomyosarcoma cell line TE671, the latter being used often as a model for
skeletal muscle diseases. In the primary skeletal muscle cells, statins alone not only
down-regulated the constitutive MHC class I expression but also impaired the IFN-γ-
induced MHC class I expression. In contrast, in the TE671, statins alone did not affect
the constitutive MHC class I expression, albeit they enhanced the IFN-γ-induced MHC
class I expression. Expression of MHC class I in muscle fibers is a ubiquitous feature of
inflammatory myopathies, and it may be a diagnostic marker for inflammatory
myopathies (Sundaram et al. 2008). MHC class I is not expressed on the sarcolemma
of normal muscle fibers. Its presence has been reported as a marker of immune
activation, since it is involved in antigen recognition by CD8+ T cells (Hohlfeld and
Engel 1994). Transgenic mice that display up-regulation of MHC class I expression
develop a self sustaining autoimmune myositis, supporting the role of MHC class I in the
pathogenesis of myositis. Nonetheless, Confalonieri et al failed to find any T-cell
cytotoxicity accompanied with up-regulated MHC class I expression in their
dysferlinopathic patients (Confalonieri et al. 2003). However, a higher MHC class I
expression has also been found in muscular dystrophies that lack the dysferlin protein
(Confalonieri et al. 2003). Therefore it may be that the induction of MHC class I is a
Discussion
71
general phenomenon in muscle cells affected by different etiologies. Our data show that
statins alone are not able to induce MHC class I in primary muscle cells. The results
also suggest that TE671 may not be an ideal model of skeletal muscle cells, since the
cell line behaves in our study more like a tumor cell as has been seen in other tumor
cell lines (Tilkin-Mariame et al. 2005). In the murine B16F10 melanoma cell line and in
human melanoma cell lines, these authors demonstrated that statins and
geranylgeranyl transferase inhibitors enhance IFN-γ induced expression of MHC class I
antigen.
Additionally, the effect of statins was also tested on different gene products associated
with MHC class I surface expression, namely TAP1 and 2 and LMP 2 and 7. IFN-γ
treatment increased the expression of all four genes. No substantial change could be
detected in response to statin treatment; nevertheless, all three statins augmented a
further two times increase over IFN-γ induction of TAP1 expression in primary muscle
cells but not in TE671. In the latter cells, it seems that increased MHC class I
expression induced by the combination of statins + IFN-γ treatment is not due to a
reinforcement of the up-regulation of TAP and LMP genes by statins, because I was
able to demonstrate the same level of expression as by IFN-γ treatment alone.
In the primary muscle cells, statins increased the IFN- γ-induced mRNA expression of
MHC class I, TAP and LMP genes, however the surface expression of MHC I was
reduced. Since the proteins of the antigen-presenting machinery (TAP1/2 and LMP2/7)
are also induced, the negative effect on the MHC I surface expression could be caused
by disturbances in lipid raft formations or an activated control pathway on the protein
level. The latter hypothesis is supported by the observation of an increased MHC I
expression in patients with dysferlinopathies and dysferlin-deficient mice (Kostek et al.
2002; Confalonieri et al. 2003). In the TE671 rhabdomyosarcoma cells, this mechanism
may be defective as a result of the dedifferentiation of the cell. As reported above,
tumor cell lines in general seem to behave differently to statin treatment (Tilkin-Mariame
et al. 2005).
Our data show a different effect of statins in vitro in comparison to previous reported in
Discussion
72
vivo studies, by showing increased MHC class I expression in muscle biopsies from
patients with statin-induced myopathies. This might be due to the presence of other
cytokines in vivo which are absent in our in vitro system. Our findings are partially in
agreement with the previous report that statin treatment led to the development of
myositis, which was reported in association with an increased MHC class I expression
on muscle cells (Needham et al. 2007). Conceivably, the presence of other players like
IFN-γ or a different genetic background like association with certain HLA types might
also be crucial to the development of statins–induced myopathies. In idiopathic
inflammatory myopathies polymorphisms of the IFN-gamma gene have been reported
to be a risk factor for the development of myositis. This altered IFN-gamma may have
different effects in the MHC expression in patients with myositis and also statin-induced
myopathy (Chinoy et al. 2007 b). Recently, a polymorphism in the SLCO1B1 gene,
which codes for an organic anion-transporting polypeptide family member (OATP1B1)
was shown to be associated with an increased risk for development of statin-induced
myopathy (Link et al. 2008). This polymorphism obviously increases the plasma
concentration of statins (Pasanen et al. 2007) but there are no data to link the function
of OATP1B1 and the MHC class I expression. Additionally the observed effect of the
statins on MHC class I expression were not dose-dependent in our study. Investigations
about the effect of statins on MHC class I molecules seem to be all the more important,
since statins are the leading therapeutic regimen for the treatment of cardiovascular
disease (Greenwood et al. 2006) and recently have been contemplated to be used as a
novel immunomodulator. Future studies should therefore attempt to investigate cytokine
regulation in patients with statin-induced myopathies and to establish the role of MHC I
expression on muscle cell lines.
Taken together this data establish that the PM and DM patients have surface binding
autoantibodies, which show cytotoxic effects towards endothelial cells and affect the
Ca2+ influx in these cells as well. Interestingly statin treatment of muscle cells seem to
increase the immunogenicity of muscle cells, shown by an increased IgG binding of
myositis sera. It proves that the autoantibodies in PM and DM patients do have
functional role in the development of these diseases. Further, above data also show that
statins alone reduced the expression of MHC I in SKMC and had no effect on MHC I in
Discussion
73
TE671. Statins potentiated the MHC I-inducing effect of IFN-gamma in TE671, but not in
SKMC, neither on the protein level, nor on mRNA level. This leads to the conclusion
that the increased muscle MHC I expression in statin-induced myopathy might not be
induced directly by statins themselves.
Summary
74
8. Summary
Polymyositis and Dermatomyositis are both inflammatory muscle diseases. Although
different autoantibodies have been reported in DM/PM, pure muscle specific antigens
are rarely known.
The current study focuses on identification of proteins, responding to antibodies from
inflammatory myositis and screens those proteins using proteomic approach. At the
same time the functional effects of the specific autoantibodies has also been
investigated by using cytotoxicity assay and calcium imaging.
Flow cytometric antibody binding showed that myositis patients have autoantibodies
against surface epitopes of muscle cells but not to endothelial cells. As statins have
been implicated in inducing myopathy with increased expression of MHC class I in
muscle cells, statin treated cells have been analysed for surface binding of patient sera.
Patients’ sera showed increased binding to mevastatin and simvastatin treated primary
muscle cells but not to rhabdomyosarcoma cells. The 2D approach has been used to
identify muscle specific autonantigens but we could not find any specific autoantigens.
Interestingly the purified IgG from myositis patients showed cytotoxic effects against
endothelial cells but not to muscle cells. In further assessing the functional effects of
autoantibodies It has been found that the purified patients’ IgG but not control IgG
changed Ca2+ influx in endothelial cells. When analysed for statins induced changes in
MHC class I expression and other proteins (TAP and LMP) at protein and RNA level,
statins alone surprisingly reduced the expression of MHC I in SKMC and had no effect
on MHC I in TE671. Statins potentiated the MHC I-inducing effect of IFN-gamma in
TE671, but not in SKMC, neither on the protein level, nor on mRNA level. This leads to
the conclusion that the increased muscle MHC I expression in statin-induced myopathy
might not be induced directly by statins themselves. The increased binding of myositis
sera to stain treated muscle cells and to primary muscle cells and the functional effects
of these autoantibodies point to a pathogenic role of the humoral immune system in
inflammatory muscle diseases.
Zusammenfassung
75
9. Zusammenfassung
Polymyositis und Dermatomyositis sind Erkrankungen aus dem Formenkreis der
entzündlichen Muskelerkrankungen. Obwohl verschiedene Autoantikörper bei diesen
Erkrankungen beschrieben wurden, sind Muskel- oder Endothel-spezifische
Autoantikörper kaum bekannt.
In der vorliegenden Studie wurden Autoantikörper und deren Spezifität mit einem
Proteomansatz und ihre möglichen funktionellen Effekte mit Zytotoxizitäts- und
Calciumfluxmessungen bei entzündlichen Muskelerkrankungen untersucht.
Mit Hilfe der Durchflusszytometrie konnte gezeigt werden, dass Myositispatienten
Autoantikörper gegen Oberflächenepitope von Muskelzellen, aber nicht Endothelzellen
haben. Eine Behandlung von Muskelzellen mit Statinen, die vereinzelt Myositiden
erzeugen können, führt zu einer erheblichen Zunahme der Bindung von Myositis IgG an
primäre Muskelzellkulturen, aber nicht an die Rhabdomyosarkomzelllinie TE671. Die
2D-Gelelektrophorese mit anschließendem Blot führte nicht zur Identifizierung
entsprechender muskel-spezifischer Autoantigene. Interessanterweise führte Myositis
IgG zu zytotoxischen Effekten bei Endothelzellen, nicht jedoch bei Muskelzellen.
Myositis IgG-Fraktionen, aber nicht IgG von Kontrollen beeinflussten auch den
Calciuminflux in Endothelzellen.
Aufgrund der Beobachtung, dass bei Statin-induzierten Myopathien MHC Klasse I auf
Muskelzellen erhöht gefunden wurde, untersuchte ich den Einfluss von Statinen auf die
MHC Klasse I Expression in vitro. Dabei zeigte sich, dass Statine sogar zu einer
Verminderung der MHC I Expression in primären Muskelzellen (SKMC) führen, bei
TE671 Zellen war kein Effekt nachweisbar. Während Statine die Interferon-γ induzierte
MHC I Expression auf der Muskeltumorzelllinie TE671 potenzierten, wurde die MHC I
Expression auf SKMC vermindert. Dies ließ sich auf Protein- und mRNA Level zeigen,
was zu der Schlussfolgerung führt, dass die MHC I Expression bei diesen Myopathien
kein direkter Effekt der Statine ist.
References
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Die von uns nachgewiesenen Antikörper gegen Oberflächenepitope, insbesondere auch
die verstärkte Bindung an Statin-behandelte Muskelzellen, und die funktionellen Effekte
der Autoantikörper legen eine pathogene Rolle des humoralen Immunsystems bei
inflammatorischen Muskelerkrankungen nahe.
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Acknowledgements
96
Acknowledgements
My first and foremost acknowledgement goes to my principle investigator, Prof. Dr. Franz Blaes.
I am greatly indebted to him for his immense belief in me and his all time support and
encouragement during the bad and good times of my PhD career. I have started my research
career in his lab and it is only because of his able guidance, that I have reached this phase of my
PhD career. “Thank you” is a very small word to extend my gratitude to Franz.
I extend my sincere gratitude to Prof. Dr. who readily agreed to be my principle investigator and
who guided me thorugh the process of obtaining my PhD.
I sincerely thank Prof. Dr. K. T. Preissner and Dr. Sigrid Schmidt without whose co-operation;
the proteomics study would not have been possible and also to Dr. Thomas Noll and Dr.
Mohammad Arshad for their help with calcium imaging.
I would like to extend my sincere thanks to the ‘’Giessen Graduate College of Life Sciences’’
(GGL), where I had the opportunity to acquire scientific knowledge as well as soft skills.
I sincerely thank Danielle Kohr, who is a dear friend as well as my fellow colleague, for all her
encouragement and support especially during hard times of my PhD career. She has been a
source of continuous motivation for me. I would like to thank Dr. Tobias Thomas, who is more a
friend than my post-Doc and from whom I have learnt the fine aspects of research. I always
appreciate our creative discussions during my PhD work.
I would like to thank my lab buddies at Neurochemistry lab (Nina, Oliver, Diana, Sandra, Dr.
Antje Kirsten, Dr. Marlene Tschernatsch and Dr. Stefanie Altenkämper) for their all time help.
Baking X-mas cookies at Steffi’s place has been a great joy. I also would like to thank my lab
technicians, Helga, Edith, Cornelia and Marita. Thanks to Eva for checking the references.
My friends outside the lab, I am incredibly indebted to you all, especially Poornima for her help
during the last days of my thesis.
My grandmother has always been my pillar of support and it goes without saying that my
mother, my family, ever deserves my deepest gratitude.
Publications
97
Publications
1. Singh P, Kohr D, Kaps M, Blaes F. Skeletal muscle cell MHC I expression:
implications for statin-induced myopathy. Muscle Nerve. 2010; 41(2):179-84.
2. Singh P, Kohr D, Kaps M, Blaes F. Influence of statins on MHC class I expression.
Ann N Y Acad Sci. 2009; 1173:746-51. Review.
3. Kohr D, Tschernatsch M, Schmitz K, Singh P, Kaps M, Schäfer KH, Diener M,
Mathies J, Matz O, Kummer W, Maihöfner C, Fritz T, Birklein F, Blaes F.
Autoantibodies in complex regional pain syndrome bind to a differentiation-
dependent neuronal surface autoantigen. Pain. 2009;143(3):246-51.
4. Sawant SV, Kiran K, Mehrotra R, Chaturvedi CP, Ansari SA, Singh P, Lodhi N, Tuli
R. A variety of synergistic and antagonistic interactions mediated by cis-acting DNA
motifs regulate gene expression in plant cells and modulate stability of the
transcription complex formed on a basal promoter. J Exp Bot. 2005; 56(419):2345-
53.